Development of the iCubate Molecular Diagnostic Platform Utilizing Amplicon Rescue Multiplex Polymerase Chain Reaction.

We utilized Amplicon-Rescue Multiplex PCR (ARM-PCR) and microarray hybridization to develop and validate the iC-GPC Assay, a multiplexed, in vitro diagnostic test that identifies five of the most common gram positive bacteria and three clinically relevant resistance markers associated with bloodstream infections (BSI). The iC-GPC Assay is designed for use with the iC-System™, which automates sample preparation, ARM-PCR, and microarray detection within a closed cassette. Herein, we determined the limit of detection for each of the iC-GPC Assay targets to be between 3.0 × 105-1.7 × 107 CFU/mL, well below clinically relevant bacterial levels for positive blood cultures. Additionally, we tested 106 strains for assay inclusivity and observed a target performance of 99.4%. 95 of 96 non-target organisms tested negative for cross-reactivity, thereby assuring a high level of assay specificity. Overall performance above 99% was observed for iC-GPC Assay reproducibility studies across multiple sites, operators and cassette lots. In conclusion, the iC-GPC Assay is capable of accurately and rapidly identifying bacterial species and resistance determinants present in blood cultures containing gram positive bacteria. Utilizing molecular diagnostics like the iC-GPC Assay will decrease time to treatment, healthcare costs, and BSI-related mortality.

Lab-on-a-Drone: Toward Pinpoint Deployment of Smartphone-Enabled Nucleic Acid-Based Diagnostics for Mobile Health Care.

We introduce a portable biochemical analysis platform for rapid field deployment of nucleic acid-based diagnostics using consumer-class quadcopter drones. This approach exploits the ability to isothermally perform the polymerase chain reaction (PCR) with a single heater, enabling the system to be operated using standard 5 V USB sources that power mobile devices (via battery, solar, or hand crank action). Time-resolved fluorescence detection and quantification is achieved using a smartphone camera and integrated image analysis app. Standard sample preparation is enabled by leveraging the drone's motors as centrifuges via 3D printed snap-on attachments. These advancements make it possible to build a complete DNA/RNA analysis system at a cost of ∼$50 ($US). Our instrument is rugged and versatile, enabling pinpoint deployment of sophisticated diagnostics to distributed field sites. This capability is demonstrated by successful in-flight replication of Staphylococcus aureus and λ-phage DNA targets in under 20 min. The ability to perform rapid in-flight assays with smartphone connectivity eliminates delays between sample collection and analysis so that test results can be delivered in minutes, suggesting new possibilities for drone-based systems to function in broader and more sophisticated roles beyond cargo transport and imaging.

Seasonal variation of respiratory pathogen colonization in asymptomatic health care professionals: A single-center, cross-sectional, 2-season observational study.

BACKGROUND: The purpose of this study was to determine the seasonal variance of potentially pathogenic bacterial and viral organisms in nasopharyngeal specimens obtained from asymptomatic health care professionals (HCPs) during the 2014 winter and summer months. METHODS: Nasopharyngeal specimens from 100 HCPs were collected from Huntsville Hospital (Huntsville, AL) during the winter and from 100 HCPs during the summer. All subjects were tested for 22 viruses and 19 bacteria using Target Enriched Multiplex Polymerase Chain Reaction. Both seasonal cohorts were composed of students, nurses, physicians, and residents. RESULTS: Of the 100 HCPs tested during the winter, 34 subjects were colonized with at least 1 bacterium, and 11 tested positive for at least 1 virus. Methicillin-resistant Staphylococcus aureus (MRSA), Moraxella catarrhalis, and coronavirus were the most frequently detected potentially infectious agents. Of the 100 HCPs tested during the summer, 37 tested positive for at least 1 bacterium, and 4 tested positive for a viral agent. The most prevalent bacteria were MRSA and Klebsiella pneumonia. CONCLUSION: Nasopharyngeal carriage among asymptomatic HCPs was common, but the frequency and presence of potential pathogens varied with each season. Understanding the colonization and infection potential of upper respiratory organisms is important, particularly for viruses. Although asymptomatic HCPs certainly harbor a number of different potentially infectious agents, future studies are needed to determine whether colonized pathogens are transmitted or initiate infection in at-risk patient populations.

The Effect of Faster Reporting Speed for Imaging Studies on the Number of Misses and Interpretation Errors: A Pilot Study.

PURPOSE: The purpose of the study was to determine if increasing radiologist reading speed results in more misses and interpretation errors. METHODS: We selected a sample set of 53 abdomen-pelvis CT scans of variable complexity performed at a teaching hospital during the study period. We classified the CT scans into 4 categories based on their level of difficulty, with level 4 representing the most-complex cases. Five attending radiologists participated in the study. We initially established an average baseline reporting time for each radiologist. Radiologists were randomly assigned a set of 12 studies, of varying complexity, to dictate at their normal speed, and a separate set of 12 studies, of similar complexity, to read at a speed that was twice as fast as their normal speed. The major and minor misses were recorded and analyzed. A χ(2) analysis was used to compare the results. RESULTS: Reading at the faster speed resulted in more major misses for 4 of the 5 radiologists. The total number of major misses for the 5 radiologists, when they reported at the faster speed, was 16 of 60 reported cases, versus 6 of 60 reported cases at normal speed; P = .032. The average interpretation error rate of major misses among the 5 radiologists reporting at the faster speed was 26.6%, compared with 10% at normal speed. CONCLUSIONS: Our pilot study found a significant positive correlation between faster reading speed and the number of major misses and interpretation errors.

DNA polymerase preference determines PCR priming efficiency.

BACKGROUND: Polymerase chain reaction (PCR) is one of the most important developments in modern biotechnology. However, PCR is known to introduce biases, especially during multiplex reactions. Recent studies have implicated the DNA polymerase as the primary source of bias, particularly initiation of polymerization on the template strand. In our study, amplification from a synthetic library containing a 12 nucleotide random portion was used to provide an in-depth characterization of DNA polymerase priming bias. The synthetic library was amplified with three commercially available DNA polymerases using an anchored primer with a random 3' hexamer end. After normalization, the next generation sequencing (NGS) results of the amplified libraries were directly compared to the unamplified synthetic library. RESULTS: Here, high throughput sequencing was used to systematically demonstrate and characterize DNA polymerase priming bias. We demonstrate that certain sequence motifs are preferred over others as primers where the six nucleotide sequences at the 3' end of the primer, as well as the sequences four base pairs downstream of the priming site, may influence priming efficiencies. DNA polymerases in the same family from two different commercial vendors prefer similar motifs, while another commercially available enzyme from a different DNA polymerase family prefers different motifs. Furthermore, the preferred priming motifs are GC-rich. The DNA polymerase preference for certain sequence motifs was verified by amplification from single-primer templates. We incorporated the observed DNA polymerase preference into a primer-design program that guides the placement of the primer to an optimal location on the template. CONCLUSIONS: DNA polymerase priming bias was characterized using a synthetic library amplification system and NGS. The characterization of DNA polymerase priming bias was then utilized to guide the primer-design process and demonstrate varying amplification efficiencies among three commercially available DNA polymerases. The results suggest that the interaction of the DNA polymerase with the primer:template junction during the initiation of DNA polymerization is very important in terms of overall amplification bias and has broader implications for both the primer design process and multiplex PCR.

Co-detection of pandemic (H1N1) 2009 virus and other respiratory pathogens.

From May through October 2009, a total of 10,624 clinical samples from 23 US states were screened for multiple respiratory pathogen gene targets. Of 3,110 (29.3%) samples positive for pandemic (H1N1) 2009 virus, 28% contained ≥ 1 other pathogen, most commonly Staphylococcus aureus (14.7%), Streptococcus pneumoniae (10.2%), and Haemophilus influenzae (3.5%).

Cerebellar ataxia following prolonged use of metronidazole: case report and literature review.

Cerebellar toxicity is a rare adverse event in patients treated with metronidazole. Here, we present a patient who developed cerebellar toxicity accompanied by objective abnormalities on magnetic resonance imaging, and review the literature on this unusual reaction. Discontinuation of metronidazole almost always results in resolution of symptoms and structural lesions.

Diffusion-tensor MR imaging of intracranial neoplasia and associated peritumoral edema: introduction of the tumor infiltration index.

PURPOSE: To determine whether diffusion-tensor magnetic resonance (MR) imaging metrics of peritumoral edema can be used to differentiate intra- from extraaxial lesions, metastatic lesions from gliomas, and high- from low-grade gliomas. MATERIALS AND METHODS: In this study, diffusion-tensor MR imaging was performed preoperatively in 40 patients with intracranial neoplasms, including meningiomas, metastatic lesions, glioblastomas multiforme, and low-grade gliomas. Histograms of mean diffusivity (MD) and fractional anisotropy (FA) were used to analyze both the tumor and the associated T2 signal intensity abnormality. An additional metric, the tumor infiltration index (TII), was evaluated. The TII is a measure of the change in FA presumably caused by tumor cells infiltrating the peritumoral edema. Student t test and least-squares linear regression analyses were performed. RESULTS: Peritumoral MD and FA values indicated no statistically significant difference between intra- and extraaxial lesions or between high- and low-grade gliomas. Regarding intraaxial tumors, the measured mean peritumoral MD of metastatic lesions, 0.733 x 10(-3) mm(2)/sec +/- 0.061 (SD), was significantly higher than that of gliomas, 0.587 +/- 0.093 x 10(-3) mm(2)/sec (P <.05). There was also a statistically significant difference between the TIIs of the edema surrounding meningiomas and metastases (mean, 0 +/- 35) and the TIIs of the edema surrounding gliomas (mean, 64 +/- 59) (P <.05). CONCLUSION: Peritumoral diffusion-tensor MR imaging metrics enable the differentiation of solitary intraaxial metastatic brain tumors from gliomas. In addition, the TII enables one to distinguish presumed tumor-infiltrated edema from purely vasogenic edema.

Peritumoral diffusion tensor imaging of high-grade gliomas and metastatic brain tumors.

BACKGROUND AND PURPOSE: Diffusion tensor imaging (DTI) is an advanced MR technique that describes the movement of water molecules by using two metrics, mean diffusivity (MD), and fractional anisotropy (FA), which represent the magnitude and directionality of water diffusion, respectively. We hypothesize that alterations in these values within the tissue surrounding brain tumors reflect combinations of increased water content and tumor infiltration and that these changes can be used to differentiate high-grade gliomas from metastatic lesions. METHODS: DTI was performed in 12 patients with high-grade gliomas and in 12 with metastatic lesions. DTI measurements were obtained from regions of interest (ROIs) placed on normal-appearing white matter and on the vasogenic edema, the T2 signal intensity abnormality surrounding each tumor. RESULTS: The peritumoral region of both gliomas and metastatic tumors displayed significant increases in MD (P <.005) and significant decreases in FA (P <.005) when compared with those of normal-appearing white matter. Furthermore, the peritumoral MD of metastatic lesions measured significantly greater than that of gliomas (P <.005). Peritumoral FA measurements, on the other hand, showed no such discrepancy. CONCLUSION: When compared with an internal control, diffusion metrics are clearly altered within the vasogenic edema surrounding both high-grade gliomas and metastatic tumors, reflecting increased extracellular water. Although peritumoral MD can be used to distinguish high-grade gliomas from metastatic tumors, peritumoral FA demonstrated no statistically significant difference. The FA changes surrounding gliomas, therefore, can be attributed not only to increased water content, but also to tumor infiltration.