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Direct determination of stability of protease inhibitors in plasma by HPLC with automated column-switching.

Automated column-switching HPLC methods were developed and utilized for the direct analyses of three hydroxamic acid based metalloprotease inhibitors in rat plasma. These column-switching methods involved the use of a restricted-access media (RAM) precolumn and a column-switching valve, allowing the complete automation of sample preparation and HPLC. The plasma samples were directly injected onto a precolumn packed with SPS/ODS stationary phase and then backflushed onto an ODS analytical column using a 6-port column-switching device. The drug stability in rat plasma was determined using both the automated and traditional HPLC methods. The results obtained from the automated column-switching methods were in good agreement with those from traditional methods that involve sequential protein precipitation, liquid extraction, solvent evaporation, and sample reconstitution. In addition to the elimination of labor-intensive and time-consuming sample preparation procedures, the column-switching methods allowed on-line analyte enrichment and accurate determination of drug stability in plasma with detection limits in the range of 10-20 ng/ml(-1). This work represents, for the first time, a drug stability study in plasma by automated column-switching HPLC technique with the use of a RAM column. Our column-switching methods can be readily adapted to any existing HPLC system with minimal hardware modification.

Animals↗

Effects of the neurotoxin 3,3'-iminodipropionitrile on acoustic startle and locomotor activity in rats: a comparison of functional observational and automated startle assessment methods.

Iminodipropionitrile (IDPN) is a neurotoxin that has been used in the validation of the U.S. Environmental Protection Agency's Functional Observational Battery (FOB), including acoustic startle. We compared the FOB clicker startle method to an automated procedure. IDPN was administered IP to male Long-Evans rats in three daily doses of 0, 100, 200, or 400 mg/kg (N = 8 per group). There was a significant effect of IDPN on clicker startle that was attributable to reduced startle in the IDPN-400 group. There were multiple significant effects of IDPN on automated startle. The overall effect of IDPN on automated startle was to reduce startle amplitude in the IDPN-400 and -200 groups. In addition, the IDPN-400 group showed startle reductions on all days, whereas the IDPN-200 group showed reduced startle primarily on day 1. IDPN also significantly altered locomotor activity, which was included as an internal check on IDPN's efficacy. The typical pattern of hypolocomotion was found on day 2 posttreatment in the IDPN-400 and -200 groups, followed by hyperlocomotion on all subsequent days in the IDPN-400 group. The startle results demonstrated that automated startle is more sensitive (at least for IDPN treatment), eliminates observer judgments, and provides interval-scaled data compared to the clicker method. However, automated startle also requires additional initial cost and more testing time per animal when multiple trials are presented.

Animals↗

Application and Quantitative Validation of Computer-Automated Three-Dimensional Counting of Cell Nuclei.

: This study provides a quantitative validation of qualitative automated three-dimensional (3-D) analysis methods reported earlier. It demonstrates the applicability and quantitative accuracy of our method to detect, characterize, and count Feulgen stained cell nuclei in two tissues (hippocampus and testes). These methods can provide important insights into the interpretation of biological, pharmacological, pathological, and toxicological events. A laser-scanned confocal light microscope was used to record 3-D images in which our algorithms automatically identified individual nuclei from the optical sections given an estimate of minimum nuclear size. The hippocampal data sets were also manually counted independently by five trained observers using the STERECON 3-D image reconstruction system. The automated and manual counts were compared. The computer counts were lower ( approximately 14%) than the manual counts, mainly because the algorithms counted a nucleus only if it was present in five consecutive optical sections but the human counters included nuclei that were in fewer optical sections. A nucleus-by-nucleus comparison of the manual and automated counts verified that the automated analysis was accurate and reproducible, and permitted additional quantitative analyses not available from manual methods. The algorithms also identified subpopulations of nuclei within the hippocampal samples, and haploid and diploid nuclei in the testes. Our methods were shown to be repeatable, accurate, and more consistent than manual counting. Nuclei in regions of high (hippocampal pyramidal layer) and low (extrapyramidal layer) density were distinguished with equal ease. Haploid and diploid nuclei were distinguished in the testes, demonstrating that our automated method may be useful for ploidy analysis. The results presented here on hippocampus and testis are consistent with other qualitative results from the liver and from immunohistochemically labeled substantia nigra, demonstrating the applicability of our software across tissues and preparation methods.

Journal Article↗

Quantitative determination of noncovalent binding interactions using automated nanoelectrospray mass spectrometry.

Electrospray ionization mass spectrometry (ESI-MS) has proven to be an extremely powerful tool for studying biomolecular structures and noncovalent interactions. Here we report a method using a fully automated, chip-based nanoESI-MS system to determine the dissociation constants (Kd) for the complexes of two different proteins with their ligands. The automated nanoelectrospray system, consisting of the NanoMate and ESI chip, serves functionally as a combination of autosampler and nanoelectrospray ionization source. This system provides all the advantages of conventional nanoelectrospray plus automated, high-throughput analyses without carryover. The automated nanoESI system was used to investigate quantitative noncovalent interactions between ribonuclease A (RNase A) and cytidylic acid ligands (2'-CMP, CTP), a well-characterized model protein-ligand complex, and between an inactive endocellulase mutant (Thermobifida fusca Cel6A D117Acd) and four oligosaccharide ligands (cellotriose, cellotetraose, cellopentaose, cellohexaose). Both titration and competitive binding approaches were performed prior to automated nanoESI-MS analysis with a Q-TOF mass spectrometer. Dissociation constants for each complex were calculated from the sum of ion peak areas of free and complexed proteins during the titration and competition experiments. The measured Kd values for the RNase A-CMP and Cel6A D117Acd-G3 complexes were found to be in excellent agreement with the available published values obtained by standard spectroscopic titration techniques. To our knowledge, this is the first report of using an ESI-MS approach to study the interactions between a cellulase and oligosaccharides. The results provide new insights for understanding the nature of cellulase-cellulose interactions.

Animals↗

Automation of a surface sampling probe/electrospray mass spectrometry system.

An image analysis automation concept and the associated software (HandsFree TLC/MS) were developed to control the surface sampling probe-to-surface distance during operation of a surface sampling electrospray system. This automation system enables both "hands-free" formation of the liquid microjunction used to sample material from the surface and hands-free reoptimization of the microjunction thickness during a surface scan to achieve a fully automated surface sampling system. The image analysis concept and the practical implementation of the monitoring and automated adjustment of the sampling probe-to-surface distance (i.e., liquid microjunction thickness) are presented. The added capabilities for the preexisting surface sampling electrospray system afforded through this software control are illustrated by an example of automated scanning of multiple development lanes on a reversed-phase C8 TLC plate and by imaging inked lettering on a paper surface. The post data acquisition processing and data display aspects of the software package are also discussed.

Chromatography, Thin Layer↗

Automated RNA selection.

In vitro selection can be used to generate nucleic acid ligands (aptamers) to target molecules ranging in size and structure from cations to cells. However, the selection process is repetitive and time-consuming. We have automated a protocol for in vitro selection using an augmented Beckman Biomek 2000 pipetting robot. The automated selection procedure requires the integration of four devices and the optimization of four molecular biology methods, and is one of the most complex automated protocols attempted to date. Initial attempts at selection yielded robust replication parasites, but optimization of the automated selection procedure suppressed the emergence of these parasites and led to the selection of true nucleic acid ligands. Automated selection can now be used to generate nucleic acid aptamers in days rather than weeks or months.

Base Composition↗

Systolic blood pressure at end-expiration measured by the automated systolic pressure variation monitor is equivalent to systolic blood pressure during apnea.

OBJECTIVE: It is necessary to define a reference systolic arterial blood pressure (RP) to calculate delta-Up (dUp) and delta-Down (dDown) for systolic pressure variation. Most studies define the reference pressure as the average systolic blood pressure during a short period of apnea. We describe an automated systolic pressure variation monitor that measures airway pressure and defines the reference pressure as the systolic blood pressure at end-expiration. The present study compares the reference systolic blood pressure measured at end-expiration by the automated systolic pressure variation monitor and the reference systolic blood pressure measured during apnea to test whether the end-expiration value is an adequate substitute for the value during apnea. METHODS: After obtaining informed consent, 108 sets of measurements of systolic pressure variation (SPV) were made in 20 intubated, mechanically-ventilated, anesthetized patients by the automated SPV monitor and during apnea. Measurements were taken during periods of hemodynamic stability defined as three consecutive end-expiratory systolic blood pressures within four mmHg of each other. The three systolic pressures at end-expiration were averaged (RPmonitor). Immediately following these measurements, the ventilator was turned off and the systolic blood pressure was measured at 6, 8, 10 and 12 seconds of apnea. The reference pressure during apnea (RPapnea) was defined as the average of the systolic blood pressure at 8, 10 and 12 seconds of apnea. For each measurement set, RPmonitor and the systolic blood pressure at 6 seconds of apnea (SBP6) were compared to RPapnea using Bland-Altman analysis. RESULTS: Bland-Altman analysis for the difference between SBP6 and RPapnea yielded a small bias of -0.3 mmHg with standard deviation of 1.3, indicating that the systolic pressure tends to continue to increase slightly after 6 seconds of apnea. Results were similar for the difference between RPmonitor and RPapnea (-0.2 +/- 3.1 mmHg). CONCLUSIONS: dUp and dDown are calculated using the reference pressure. RPmonitor is an average of 0.2 mm Hg less than RPapnea, thus dUp calculated by the automated SPV monitor is an avenge of 0.2 mm Hg greater than dUp measured by the reference pressure during apnea and dDown is 0.2 mm Hg less. Since the bias of -0.2 mmHg is clinically insignificant, there is acceptable agreement between the reference pressure obtained during apnea and that obtained by the automated SPV monitor at end-expiration. The mean difference between RPmonitor and RPapnea is explained by the continued rise in systolic pressure during the period of apnea as demonstrated by the difference between SBP6 and RPapnea.

Anesthesia↗

End-diastolic and end-systolic volume from the left ventricular angiogram: how accurate is visual frame selection? Comparison between visual and semi-automated comnputer-assisted analysis.

BACKGROUND: End-diastolic (ED), end-systolic (ES) left ventricular (LV) volumes and LV ejection fraction (LVEF) are important parameters for clinical decision making in heart disease. In clinical practice the frames from cine-angiography with the largest and smallest opacified LV areas are visually selected and the endocardial borders traced as LVED and LVES contours, respectively. We compared the accuracy of this visual method using two frames with a semi-automated computer assisted frame-by-frame analysis of the complete opacified cardiac cycles. METHODS AND RESULTS: In 17 patients a biplane LV cine-angiogram was obtained at 25 frames/s. Complete frame-by-frame analysis was performed using semi-automatic border detection software. Experienced independent observers visually selected and manually traced LVED and LVES in the so-called visually assessed two-frame method in a consensus meeting. LV volumes were calculated by the area-length method. Mean LVEDV, LVESV and LVEF were 133 +/- 57, 56 +/- 40 ml and 61 +/- 16%, respectively, for the visually assessed two-frame method, and 117 +/- 49, 53 +/- 33 ml and 60 +/- 13%, respectively, for the semi-automated computer assisted frame-by-frame method. LVEDV was significantly higher in the visually assessed two-frame method (p < 0.01). Linear regression analysis showed an excellent correlation between semi-automated computer-assisted frame-by-frame and the visually assessed two-frame LVEDV (y = 1.2x - 2.9; r2 = 0.98), LVESV (y = 1.2x - 8.2; r2 = 0.97) and good linear correlation for LVEF (p = 1.2x - 3.6; r2 = 0.82). Bland-Altman analysis showed respectively a bias of 16.4, 2.4 ml and 5.0% with overall wide limits of agreement (-6.6 and 39.4 ml; -16.6 and 21.4 ml; -9.0% and 19.1%). CONCLUSION: Correlation is excellent when visually assessed LVED and LVES are compared with a semi-automated computer assisted frame-by-frame analysis. However, the visually assessed two-frame method tends to overestimate the volumes obtained by semi-automated computer-assisted frame-by-frame analysis, especially for LVEDV, indicating that visual selection will yield a higher LVEF, which may influence clinical decision making.

Angiocardiography↗

An automated approach to salt selection for new unique trazodone salts.

PURPOSE: The purpose of this study was to establish an automated approach to salt selection and to search for unique trazodone salts for new applications. METHODS: Automated procedures were developed on a Biomek 2000 automation workstation with stacker and plate reader capabilities. Trazodone was dispensed into 96-well plates, and an automated method was set up to form 104 trazodone salts. Salts were observed under a polarized light microscope to determine crystallinity. After stepwise eliminations, the remaining salts were scaled-up and subjected to differential scanning calorimetry (DSC), powder x-ray diffraction (PXRD), hygroscopic, pH-solubility, density, surface area, and particle size analyses. RESULTS: Oils formed in several cases resulting in preliminary elimination of mesyl and esyl salts and four crystallizing solvents. Crystallinity was observed in 34 of 44 scaled-up trazodone salts. PXRD, DSC, and hygroscopic analyses indicated a number of new salts that were comparable in physicochemical parameters to the marketed HCl salt. Among them, the tosylate salt showed uniqueness for new applications. CONCLUSIONS: Automated procedures can be developed to increase the efficiency of pharmaceutical salt selection. The new tosylate salt gave a unique pH-solubility profile with low solubility over the entire pH range making it a potential candidate for a suspension or prolonged action formulation.

Hydrogen-Ion Concentration↗

Automated screening for genomic imbalances using matrix-based comparative genomic hybridization.

Genome-wide screening for chromosomal imbalances using comparative genomic hybridization (CGH) revealed a wealth of data on previously unrecognized tumor-specific genomic alterations. CGH to microarrays of DNA, an approach termed matrix-CGH, allows detection of genomic imbalances at a much higher resolution. We show that matrix CGH is also feasible from small tissue samples requiring universal amplification of genomic DNA. Because widespread application of matrix-CGH experiments using large numbers of DNA targets demands a high degree of automation, we have developed a protocol for a fully automated procedure. The use of specialized instrumentation for the generation of DNA chips, their hybridization, scanning, and evaluation required numerous alterations and modifications of the initial protocol. We here present the elaboration and testing of automated matrix-CGH. A chip consisting of 188 different genomic DNA fragments, cloned in bacterial artificial chromosome (BAC) or P1-derived artificial chromosome (PAC) vectors and immobilized in replicas of 10, was used to assess the performance of the automated protocol in determining the gene dosage variations in tumor cell lines COLO320-HSR, HL60, and NGP. Although ratios of matrix-CGH were highly concordant with results of chromosomal CGH (85%), the dynamic range of the matrix-CGH ratios was highly superior. Investigation of the two amplicons on 8q24 in COLO320-HSR and HL60, containing the MYC gene, revealed a homogeneous amplicon in COLO320-HSR but a heterogeneous amplification pattern in HL60 cells. Although control clones for normalization of the signal ratios can be predicted in cases with defined chromosomal aberrations, in primary tumors such data are often not available, requiring alternative normalization algorithms. Testing such algorithms in a primary high-grade B-cell lymphoma, we show the feasibility of this approach. With the matrix-CGH protocol presented here, robust and reliable detection of genomic gains and losses is accomplished in an automated fashion, which provides the basis for widespread application in tumor and clinical genetics.

Chromosome Aberrations↗

Enrichment of peripheral blood CD34+ cells for transplantation using a fully automated immunomagnetic cell selection system and a novel octapeptide releasing agent.

Positive selection of CD34+ cells is being increasingly performed to support hematological reconstitution following high-dose and dose-intensive chemotherapy and to reduce the non-target cell content of transplants. The present study was designed to evaluate the performance of an immunomagnetic cell selection system, including comparison of enzyme and peptide releasing agents and of semi-automated and fully automated selection systems. A total of 74 immunomagnetic CD34+ cell selection procedures were performed involving 55 subjects, the majority of whom had hematologic malignancies. Median CD34+ cell purity with a newly developed specific octapeptide releasing agent (98.5%; 81.0-99.0%) was significantly higher (P = 0.002) than that with chymopapain (85.8%; 28.1-99.7%). No significant differences were observed between semi-automated and fully automated systems in CD34+ cell purity or yield or time to WBC or platelet recovery. Immunomagnetic selection was found to provide highly purified populations of CD34+ cells in sufficient numbers for use in transplantation procedures. CD34+ cell transplants supported rapid and reliable hematologic reconstitution. Use of a fully automated system markedly reduced the time and labor required for immunomagnetic selection, potentially affording more standardized and reproducible positive selection of CD34+ cells.

Adult↗

Automated extraction and amplification of DNA from whole blood using a robotic workstation and an integrated thermocycler.

Growing knowledge of the genetic basis of inheritable diseases has resulted in a rapidly increasing demand for DNA mutation analysis. Current methods are reliable and suitable for low-throughput mutation analyses, but are unable to cope with the increasing demand for genetic analyses, necessitating the development of new, fully automated and reliable methods. We developed a semi-automated method for DNA mutation analysis by integrating a thermocycler into a robotic pipetting workstation. DNA was extracted from 84 samples of 10 microl of EDTA-treated whole blood using magnetic beads within 2 h. Directly after isolation, the DNA was automatically transferred to an integrated thermocycler for amplification. Our semi-automated method proved to be reliable and robust, showing unambiguously interpretable PCR signals without occurrence of contamination. It is also faster than conventional manual methods. Only a brief manual intervention is required to remove and refit the seal of the PCR plate. This semi-automated assay is a step forward in the development of fully automated assays for DNA mutation analysis.

DNA↗

Preparation of some immobilized linked enzyme systems and their use in the automated determination of disaccharides.

1. Glucose oxidase (EC 1.1.3.4), amyloglucosidase (EC 3.2.1.3), invertase (EC 3.2.1.26) and beta-galactosidase (EC 3.2.1.23) were covalently attached via glutaraldehyde to the inside surface of nylon tube. 2. The linked enzyme system, comprising invertase immobilized within a nylon tube acting in series with glucose oxidase immobilized in a similar way, was used for the automated determination of sucrose. 3. The linked enzyme system, comprising beta-galactosidase immobilized within a nylon tube acting in series with glucose oxidase immobilized in a similar way, was used for the automated determination of lactose. 4. The linked enzyme system, comprising amyloglucosidase immobilized within a nylon tube acting in series with glucose oxidase immobilized in a similar way, was used for the automated determination of maltose. 5. Mixtures of glucose oxidase and amyloglucosidase were immobilized within the same piece of nylon tube and used for the automated determination of maltose. 6. Mixtures of glucose oxidase and invertase were immobilized within the same piece of nylon tube and used for the automated determination of sucrose.

Aspergillus↗

Automated counting of white and red blood cells in the cerebrospinal fluid.

The objective of this study was to examine to what extent the automated method of the Bayer H*2 instrument could replace the visual counting of white and red blood cells in cerebrospinal fluid. The number of white blood cells as well as the percentage of mononuclear and polymorphonuclear cells were counted in the 'Baso channel' (Research screen 3) whereas the number of red cells were registered as the 'R-count' (Research screen 1). All automated cell counts were compared to visual estimates. The automated count yielded reliable results down to 5 x 106 white blood cells/l and 5 x 108 red blood cells/l. In some samples 'noise' was present in the Baso channel. A correct white blood cell count could then be obtained by counting the cells directly as dots on the screen. It was possible to differentiate between polymorphnuclear cells and mononuclear cells at all WBC concentrations. The automated counting of cerebrospinal fluid can be performed without changing thresholds or sample volumes of the instrument. Thus, in the routine practice it will be possible to alternate between automated counting of whole blood samples and cerebrospinal fluid samples.

Adult↗

Software reuse reference model approach in developing an automated medical information system (AMIS) for improving health care practice.

OBJECTIVE: To evaluate patient acceptance of an automated medical information system specific to headache. BACKGROUND: Studies suggest that automated tools may help health care delivery systems to be efficient and effective, but patient satisfaction remains a major concern. METHODS: We adapted our software reuse reference model and Unified Modeling Language to apply the domain model to the headache population. Patients with headache were tested both to validate the system and to evaluate patient satisfaction and headache management with an automated system. RESULTS: The mean age of all study participants was 44 years. Over 95% of the participants were satisfied or strongly satisfied with the Automated Medical Information System. CONCLUSIONS: The results strongly suggest that patients are willing and able to use nontraditional sources, such as the Automated Medical Information System, to learn about their illnesses.

Adult↗

[Automated swinging flashlight test in patients with optic nerve diseases].

AIM OF THE STUDY: The swinging flashlight test is an objective method to diagnose a lesion of the anterior visual pathways. However, errors and faults may easily alter the test's results. Hence, the value of the swinging flashlight test depends highly on the examiner's skills. Therefore an automated and objective procedure was developed which is independent from the examiner. METHODS: A binocularly measuring instrument adapted for video pupillography was supplied with two arrays of light emitting diodes in front of each eye of the subject. By means of this illumination, pupillary light reflexes are elicited alternately. Pupil size is registered continuously, and after artifact elimination, the response amplitudes of the pupils are determined as a mean of right and left pupil. Responses elicited via right and left eye are compared. By varying the stimulus intensity it is possible to measure the amount of the relative afferent pupillary defect. The procedure was tested in 31 patients with optic nerve disorders. RESULTS: The measurements were easily feasible, stable and reliable. Correlation between the relative afferent pupillary defect detected manually by grey filter compensating and with the automated procedure proved to be high. Both variables correlated highly significant with a Spearman rank coefficient of 0.65. If the clinical test is regarded as the golden standard, the automated swinging flashlight test is able to detect 85% of the relative afferent pupillary defects > or = 0.3 logE and 94% of the defects > or = 0.6 logE. CONCLUSION: The automated swinging flashlight test can be recommended to exclude influences by the examiner or if the exact amount of the relative afferent pupillary defect is desired, e.g. when monitoring therapeutic effects in optic nerve diseases. Furthermore, an automated swinging flashlight test could serve as a screening test.

Adult↗

An automated robotic approach with redundant navigation for minimal invasive extended transsphenoidal skull base surgery.

BACKGROUND: The aim of this work was to determine the feasibility of a robotic-assisted and fully automated approach to the sphenoid sinus. An image-guided robotic system was designed to address potential human errors in performing transsphenoidal sinus surgery by combining the reproducible accuracy of a robotic system with standard computer navigation. METHODS: A six-degrees of freedom robotic assistance system and an opto-electrical navigation system were combined for image-guided assistance with redundantly controlled robotics. Newly designed endoscopic instruments for robotic surgery have been developed and are described. Telemanipulatory, as well as fully automated procedures, were tested on cadaveric heads as part of a preclinical trial. RESULTS: A fully automated sphenoidotomy as well as a telemanipulatory sphenoidectomy were performed successfully on cadaveric heads. Intraoperative performance, accuracy assessment studies, as well as possible sources of stereotactic offsets are described. The mean measured robotic reproducibility accuracy was 0.056 mm (range: 0.02 - 0.14 mm) and the mean overall navigated robotic accuracy, including all transformation and registration errors was 1.53 mm (range: 1.13 - 1.89 mm) respectively. CONCLUSION: A system for robot-guided surgery in combination with redundant navigational control was developed. It allows highly accurate maneuvers, performed either in a telemanipulation mode as master-slave system or in a fully automated fashion. A sphenoidectomy on cadaveric heads was performed in both telemanipulation and fully automated modes. The overall intraoperative accuracy was in the range of the resolution of the CT images and stereotactic offsets were caused mainly due to deflections of the endoscopic operating instrument.

Cadaver↗

Preliminary assessment of an automated surveillance system for infection control.

BACKGROUND AND OBJECTIVE: Rapid identification and investigation of potential outbreaks is key to limiting transmission in the healthcare setting. Manual review of laboratory results remains a cumbersome, time-consuming task for infection control practitioners (ICPs). Computer-automated techniques have shown promise for improving the efficiency and accuracy of surveillance. We examined the use of automated control charts, provided by an automated surveillance system, for detection of potential outbreaks. SETTING: A 656-bed academic medical center. METHODS: We retrospectively reviewed 13 months (November 2001 through November 2002) of laboratory-patient data, comparing an automated surveillance application with standard infection control practices. We evaluated positive predictive value, sensitivity, and time required to investigate the alerts. An ICP created 75 control charts. A standardized case investigation form was developed to evaluate each alert for the likelihood of nosocomial transmission based on temporal and spatial overlap and culture results. RESULTS: The 75 control charts were created in 75 minutes and 18 alerts fired above the 3-sigma level. These were independently reviewed by an ICP and associate hospital epidemiologist. The review process required an average of 20 minutes per alert and the kappa score between the reviewers was 0.82. Eleven of the 18 alerts were determined to be potential outbreaks, yielding a positive predictive value of 0.61. Routine surveillance identified 5 of these 11 alerts during this time period. CONCLUSION: Automated surveillance with user-definable control charts for cluster identification was more sensitive than routine methods and is capable of operating with high specificity and positive predictive value in a time-efficient manner.

Cross Infection↗