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The class activation map (CAM) represents the neural-network-derived region of interest, which can help clarify the mechanism of the convolutional neural network's determination of any class of interest. In medical imaging, it can help medical practitioners diagnose diseases like COVID-19 or pneumonia by highlighting the suspicious regions in Computational Tomography (CT) or chest X-ray (CXR) film. Many contemporary deep learning techniques only focus on COVID-19 classification tasks using CXRs, while few attempt to make it explainable with a saliency map. To fill this research gap, we first propose a VGG-16-architecture-based deep learning approach in combination with image enhancement, segmentation-based region of interest (ROI) cropping, and data augmentation steps to enhance classification accuracy. Later, a multi-layer Gradient CAM (ML-Grad-CAM) algorithm is integrated to generate a class-specific saliency map for improved visualization in CXR images. We also define and calculate a Severity Assessment Index (SAI) from the saliency map to quantitatively measure infection severity. The trained model achieved an accuracy score of 96.44% for the three-class CXR classification task, i.e., COVID-19, pneumonia, and normal (healthy patients), outperforming many existing techniques in the literature. The saliency maps generated from the proposed ML-GRAD-CAM algorithm are compared with the original Gran-CAM algorithm.

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Predicting inpatient length of stay (LoS) is important for hospitals aiming to improve service efficiency and enhance management capabilities. Patient medical records are strongly associated with LoS. However, due to diverse modalities, heterogeneity, and complexity of data, it becomes challenging to effectively leverage these heterogeneous data to put forth a predictive model that can accurately predict LoS. To address the challenge, this study aims to establish a novel data-fusion model, termed as DF-Mdl, to integrate heterogeneous clinical data for predicting the LoS of inpatients between hospital discharge and admission. Multi-modal data such as demographic data, clinical notes, laboratory test results, and medical images are utilized in our proposed methodology with individual "basic" sub-models separately applied to each different data modality. Specifically, a convolutional neural network (CNN) model, which we termed CRXMDL, is designed for chest X-ray (CXR) image data, two long short-term memory networks are used to extract features from long text data, and a novel attention-embedded 1D convolutional neural network is developed to extract useful information from numerical data. Finally, these basic models are integrated to form a new data-fusion model (DF-Mdl) for inpatient LoS prediction. The proposed method attains the best R2 and EVAR values of 0.6039 and 0.6042 among competitors for the LoS prediction on the Medical Information Mart for Intensive Care (MIMIC)-IV test dataset. Empirical evidence suggests better performance compared with other state-of-the-art (SOTA) methods, which demonstrates the effectiveness and feasibility of the proposed approach.

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There is an urgent need to improve the diagnostic capacity of cutaneous leishmaniasis (CL) in rural health centers to improve the management of the disease in patients from remote regions where the infection is endemic. Microscopy of Giemsa-stained lesion smears is the standard-of-care diagnostic test in virtually all health centers, but its sensitivity is suboptimal (50-70%) and prone to false negative results. We evaluated the performance of a low-cost DNA extraction buffer (LAB) using a portable miniPCR™ equipment coupled with an inexpensive fluorescence viewer to detect Leishmania DNA with the naked eye or using a commercial photo app. Using ten-fold serial dilutions of Leishmania (V.) panamensis promastigotes the miniPCR-F test detected 10 parasites per µL, which was comparable to real-time PCR. Utilization of DNA from retrospective clinical samples preserved at -80 °C from Colombia (n = 28) or lesion exudate preserved in filter papers from Peru (n = 48) showed that the miniPCR-fluorescent test had a 100% sensitivity and > 90% specificity compared to real-time PCR. This study demonstrated the utility of LAB DNA extraction method for direct amplification of Leishmania using the miniPCR and reading of P51 results with the naked eye or via digital reading with a photo app. These preliminary results indicated that the miniPCR-F test workflow could be amenable to implementation in resource-limited health centers.

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Providing timely patient care while maintaining optimal resource utilization is one of the central operational challenges hospitals have been facing throughout the pandemic. Hospital length of stay (LOS) is an important indicator of hospital efficiency, quality of patient care, and operational resilience. Numerous researchers have developed regression or classification models to predict LOS. However, conventional models suffer from the lack of capability to make use of typically censored clinical data. We propose to use time-to-event modeling techniques, also known as survival analysis, to predict the LOS for patients based on individualized information collected from multiple sources. The performance of six proposed survival models is evaluated and compared based on clinical data from COVID-19 patients.

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BACKGROUND: With the emergence of continuously mutating variants of coronavirus, it is urgent to develop a deep learning model for automatic COVID-19 diagnosis at early stages from chest X-ray images. Since laboratory testing is time-consuming and requires trained laboratory personal, diagnosis using chest X-ray (CXR) is a befitting option. OBJECTIVE: In this study, we proposed an interpretable multi-task system for automatic lung detection and COVID-19 screening in chest X-rays to find an alternate method of testing which are reliable, fast and easily accessible, and able to generate interpretable predictions that are strongly correlated with radiological findings. METHODS: The proposed system consists of image preprocessing and an unsupervised machine learning (UML) algorithm for lung region detection, as well as a truncated CNN model based on deep transfer learning (DTL) to classify chest X-rays into three classes of COVID-19, pneumonia, and normal. The Grad-CAM technique was applied to create class-specific heatmap images in order to establish trust in the medical AI system. RESULTS: Experiments were performed with 15,884 frontal CXR images to show that the proposed system achieves an accuracy of 91.94% in a test dataset with 2,680 images including a sensitivity of 94.48% on COVID-19 cases, a specificity of 88.46% on normal cases, and a precision of 88.01% on pneumonia cases. Our system also produced state-of-the-art outcomes with a sensitivity of 97.40% on public test data and 88.23% on a previously unseen clinical data (1,000 cases) for binary classification of COVID-19-positive and COVID-19-negative films. CONCLUSION: Our automatic computerized evaluation for grading lung infections exhibited sensitivity comparable to that of radiologist interpretation in clinical applicability. Therefore, the proposed solution can be used as one element of patient evaluation along with gold-standard clinical and laboratory testing.

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We present a miniaturized centrifugal platform that uses density centrifugation for separation and analysis of biological components in small volume samples (~5 µL). We demonstrate the ability to enrich leukocytes for on-disk visualization via microscopy, as well as recovery of viable cells from each of the gradient partitions. In addition, we simplified the traditional Modified Wright-Giemsa staining by decreasing the time, volume, and expertise involved in the procedure. From a whole blood sample, we were able to extract 95.15% of leukocytes while excluding 99.8% of red blood cells. This platform has great potential in both medical diagnostics and research applications as it offers a simpler, automated, and inexpensive method for biological sample separation, analysis, and downstream culturing.

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Waterborne pathogens pose significant threat to the global population and early detection plays an important role both in making drinking water safe, as well as in diagnostics and treatment of water-borne diseases. We present an innovative centrifugal sedimentation immunoassay platform for detection of bacterial pathogens in water. Our approach is based on binding of pathogens to antibody-functionalized capture particles followed by sedimentation of the particles through a density-media in a microfluidic disk. Beads at the distal end of the disk are imaged to quantify the fluorescence and determine the bacterial concentration. Our platform is fast (20 min), can detect as few as ∼10 bacteria with minimal sample preparation, and can detect multiple pathogens simultaneously. The platform was used to detect a panel of enteric bacteria (Escherichia coli, Salmonella typhimurium, Shigella, Listeria, and Campylobacter) spiked in tap and ground water samples.

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Natural killer (NK) cells have innate antibacterial activity that could be targeted for clinical interventions for infectious disease caused by naturally occurring or weaponized bacterial pathogens. To determine a potential role for NK cells in immunity to Bacillus anthracis, we utilized primary human and murine NK cells, in vitro assays, and in vivo NK cell depletion in a murine model of inhalational anthrax. Our results demonstrate potent antibacterial activity by human NK cells against B. anthracis bacilli within infected autologous monocytes. Surprisingly, NK cells also mediate moderate antibacterial effects on extracellular vegetative bacilli but do not have activity against extracellular or intracellular spores. The immunosuppressive anthrax lethal toxin impairs NK gamma interferon (IFN-γ) expression, but neither lethal nor edema toxin significantly alters the viability or cytotoxic effector function of NK cells. Compared to human NK cells, murine NK cells have a similar, though less potent, activity against intracellular and extracellular B. anthracis. The in vivo depletion of murine NK cells does not alter animal survival following intranasal infection with B. anthracis spores in our studies but significantly increases the bacterial load in the blood of infected animals. Our studies demonstrate that NK cells participate in the innate immune response against B. anthracis and suggest that immune modulation to augment NK cell function in early stages of anthrax should be further explored in animal models as a clinical intervention strategy.

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Anthrax lethal toxin (LeTx) and edema toxin (EdTx) have been shown to alter hemodynamics in the rodent model, while LeTx primarily is reported to induce extensive tissue pathology. However, the rodent model has limitations when used for comparison to higher organisms such as humans. The rabbit model, on the other hand, has gained recognition as a useful model for studying anthrax infection and its pathophysiological effects. In this study, we assessed the hemodynamic effects of lethal toxin (LeTx) and edema toxin (EdTx) in the rabbit model using physiologically relevant amounts of the toxins. Moreover, we further examine the pathological effects of LeTx on cardiac tissue. We intravenously injected Dutch-belted rabbits with either low-dose and high-dose recombinant LeTx or a single dose of EdTx. The animals' heart rate and mean arterial pressure were continuously monitored via telemetry until either 48 or 72 h post-challenge. Additional animals challenged with LeTx were used for cardiac troponin I (cTnI) quantitation, cardiac histopathology, and echocardiography. LeTx depressed heart rate at the lower dose and mean arterial pressure (MAP) at the higher dose. EdTx, on the other hand, temporarily intensified heart rate while lowering MAP. Both doses of LeTx caused cardiac pathology with the higher dose having a more profound effect. Lastly, left-ventricular dilation due to LeTx was not apparent at the given time-points. Our study demonstrates the hemodynamic effects of anthrax toxins, as well as the pathological effects of LeTx on the heart in the rabbit model, and it provides further evidence for the toxins' direct impact on the heart.

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BACKGROUND: Heart failure is a critical condition that affects many people and often results from left ventricular dysfunction. Numerous studies investigating this condition have been performed using various model systems. To do so, investigators must be able to accurately measure myocardial performance in order to determine the degree of left ventricular function. In this model development study, we employ a wireless telemetry system purchased from Data Sciences International to continuously assess left ventricular function in the rabbit model. FINDINGS: We surgically implanted pressure-sensitive catheters fitted to wireless radio-transmitters into the left ventricle of Dutch-belted rabbits. Following recovery of the animals, we continuously recorded indices of cardiac contractility and ventricular relaxation at baseline for a given time period. The telemetry system allowed us to continuously record baseline left ventricular parameters for the entire recording period. During this time, the animals were unrestrained and fully conscious. The values we recorded are similar to those obtained using other reported methods. CONCLUSIONS: The wireless telemetry system can continuously measure left ventricular pressure, cardiac contractility, and cardiac relaxation in the rabbit model. These results, which were obtained just as baseline levels, substantiate the need for further validation in this model system of left ventricular assessment.

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We used ex vivo and in vivo experiments with Xenopus laevis tadpoles to examine the hypothesis that the set-point for negative feedback on pituitary thyroid-stimulating hormone (TSH) synthesis and secretion by thyroid hormones (THs) increases as metamorphosis progresses to allow for the previously documented concomitant increase in serum TH concentrations and pituitary TSH mRNA expression during this transformative process. First, pituitaries from climactic tadpoles were cultured for up to 96 h to characterize the ability of pituitary explants to synthesize and secrete TSHß in the absence of hypothalamic and circulating hormones. Next, pituitary explants from tadpoles NF stages 54-66 were exposed to physiologically-relevant concentrations of THs to determine whether stage-specific differences exist in pituitary sensitivity to negative feedback by THs. Finally, in vivo exposures of tadpoles to THs were conducted to confirm the results of the ex vivo experiments. When pituitaries from climactic tadpoles were removed from the influence of endogenous hormones, TSHß mRNA expression increased late or not at all whereas the rate of TSHß secreted into media increased dramatically, suggesting that TSH secretion, but not TSH mRNA expression, is under the negative regulation of an endogenous signal during the climactic stages of metamorphosis. Pituitaries from pre- and prometamorphic tadpoles were more sensitive to TH-induced inhibition of TSHß mRNA expression and secretion than pituitaries from climactic tadpoles. The observed decrease in sensitivity of pituitary TSHß mRNA expression to negative feedback by THs from premetamorphosis to metamorphic climax was confirmed by in vivo experiments in which tadpoles were reared in water containing THs. Based on the results of this study, a model is proposed to explain the seemingly paradoxical, concurrent rise in serum TH concentrations and pituitary TSH mRNA expression during metamorphosis in larval anurans.

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Thyroid-stimulating hormone (TSH) is an important regulator of the hypothalamic-pituitary-thyroid (HPT) axis in Xenopus laevis. To evaluate the role of this hormone on developing tadpoles, immunologically-based Western blots and sandwich ELISAs were developed for measuring intracellular (within pituitaries), secreted (ex vivo pituitary culture), and circulating (serum) amounts. Despite the small size of the tadpoles, these methods were able to easily measure intracellular and secreted TSH, and circulating TSH was measurable in situations where high levels were induced. The method was validated after obtaining a highly purified and enriched TSH sample using anti-TSH-ß antibodies conjugated to magnetic beads. Subsequent mass-spectrometric analysis of the bands from SDS-PAGE and Western procedures identified the presence of amino acid sequences corresponding to TSH subunits. The purified sample was also used to prepare standard curves for quantitative analysis. The Western and ELISA methods had limits of detection in the low nanogram range. While the majority of the developmental work for these methods was done with X. laevis, the methods also detected TSH in Xenopus tropicalis. To our knowledge this is the first report of a specific detection method for TSH in these species, and the first to measure circulating TSH in amphibians. Examples of the utility of the methods include measuring a gradual increase in pituitary TSH at key stages of development, peaking at stages 58-62; the suppression of TSH secretion from cultured pituitaries in the presence of thyroid hormone (T4); and increases in serum TSH following thyroidectomy.

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Successful treatment of inhalation anthrax, pneumonic plague and tularemia can be achieved with fluoroquinolone antibiotics, such as ciprofloxacin and levofloxacin, and initiation of treatment is most effective when administered as soon as possible following exposure. Bacillus anthracis Ames, Yersinia pestis CO92, and Francisella tularensis SCHU S4 have equivalent susceptibility in vitro to ciprofloxacin and levofloxacin (minimal inhibitory concentration is 0.03 µg/ml); however, limited information is available regarding in vivo susceptibility of these infectious agents to the fluoroquinolone antibiotics in small animal models. Mice, guinea pig, and rabbit models have been developed to evaluate the protective efficacy of antibiotic therapy against these life-threatening infections. Our results indicated that doses of ciprofloxacin and levofloxacin required to protect mice against inhalation anthrax were approximately 18-fold higher than the doses of levofloxacin required to protect against pneumonic plague and tularemia. Further, the critical period following aerosol exposure of mice to either B. anthracis spores or Y. pestis was 24 h, while mice challenged with F. tularensis could be effectively protected when treatment was delayed for as long as 72 h postchallenge. In addition, it was apparent that prolonged antibiotic treatment was important in the effective treatment of inhalation anthrax in mice, but short-term treatment of mice with pneumonic plague or tularemia infections were usually successful. These results provide effective antibiotic dosages in mice, guinea pigs, and rabbits and lay the foundation for the development and evaluation of combinational treatment modalities.

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Braun/murein lipoprotein (Lpp) is involved in inflammatory responses and septic shock. We previously characterized a Deltalpp mutant of Yersinia pestis CO92 and found that this mutant was defective in surviving in macrophages and was attenuated in a mouse inhalation model of plague when compared to the highly virulent wild-type (WT) bacterium. We performed global transcriptional profiling of WT Y. pestis and its Deltalpp mutant using microarrays. The organisms were cultured at 26 and 37 degrees Celsius to simulate the flea vector and mammalian host environments, respectively. Our data revealed vastly different effects of lpp mutation on the transcriptomes of Y. pestis grown at 37 versus 26 degrees C. While the absence of Lpp resulted mainly in the downregulation of metabolic genes at 26 degrees C, the Y. pestis Deltalpp mutant cultured at 37 degrees C exhibited profound alterations in stress response and virulence genes, compared to WT bacteria. We investigated one of the stress-related genes (htrA) downregulated in the Deltalpp mutant relative to WT Y. pestis. Indeed, complementation of the Deltalpp mutant with the htrA gene restored intracellular survival of the Y. pestis Deltalpp mutant. Our results support a role for Lpp in Y. pestis adaptation to the host environment, possibly via transcriptional activation of htrA.

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Enterotoxigenic Escherichia coli (ETEC) produces the ADP-ribosyltransferase toxin known as heat-labile enterotoxin (LT). In addition to the toxic effect of LT resulting in increases of cyclic AMP (cAMP) and disturbance of cellular metabolic processes, this toxin promotes bacterial adherence to intestinal epithelial cells (A. M. Johnson, R. S. Kaushik, D. H. Francis, J. M. Fleckenstein, and P. R. Hardwidge, J. Bacteriol. 191:178-186, 2009). Therefore, we hypothesized that the identification of a compound that inhibits the activity of the toxin would have a suppressive effect on the ETEC colonization capabilities. Using in vivo and in vitro approaches, we present evidence demonstrating that a fluorenone-based compound, DC5, which inhibits the accumulation of cAMP in intoxicated cultured cells, significantly decreases the colonization abilities of adenylyl cyclase toxin-producing bacteria, such as ETEC. These findings established that DC5 is a potent inhibitor both of toxin-induced cAMP accumulation and of ETEC adherence to epithelial cells. Thus, DC5 may be a promising compound for treatment of diarrhea caused by ETEC and other adenylyl cyclase toxin-producing bacteria.

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Anthrax edema toxin (ET), a powerful adenylyl cyclase, is an important virulence factor of Bacillus anthracis. Until recently, only a modest amount of research was performed to understand the role this toxin plays in the organism's immune evasion strategy. A new wave of studies have begun to elucidate the effects this toxin has on a variety of host cells. While efforts have been made to illuminate the effect ET has on cells of the adaptive immune system, such as T cells, the greatest focus has been on cells of the innate immune system, particularly the macrophage. Here we discuss the immunoevasive activities that ET exerts on macrophages, as well as new research on the effects of this toxin on B cells.

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Recombinant Sindbis viruses were engineered to express alternative forms of the protective antigen (PA) of Bacillus anthracis. The recombinant viruses induced PA-specific immunoglobulin G and neutralizing antibodies in Swiss Webster mice. Vaccination with the recombinant viruses induced immunity that offered some protection from a lethal Ames strain spore challenge and synergized the protective effects of ciprofloxacin.

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We employed Murine GeneChips to delineate the global transcriptional profiles of the livers, lungs, and spleens in a mouse pneumonic plague infection model with wild-type (WT) Y. pestis CO92 and its Braun lipoprotein (Deltalpp) mutant with reduced virulence. These organs showed differential transcriptional responses to infection with WT Y. pestis, but the overall host functional processes affected were similar across all three tissues. Gene expression alterations were found in inflammation, cytokine signaling, and apoptotic cell death-associated genes. Comparison of WT and Deltalpp mutant-infected mice indicated significant overlap in lipopolysaccharide- (LPS-) associated gene expression, but the absence of Lpp perturbed host cell signaling at critical regulatory junctions resulting in altered immune response and possibly host cell apoptosis. We generated a putative signaling pathway including major inflammatory components that could account for the synergistic action of LPS and Lpp and provided the mechanistic basis of attenuation caused by deletion of the lpp gene from Y. pestis in a mouse model of pneumonic plague.

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The ability to protect mice against respiratory infections with virulent Francisella tularensis has been problematic and the role of antibody-versus-cell-mediated immunity controversial. In this study, we tested the hypothesis that protective immunity can develop in mice that were given antibiotic therapy following infection via the respiratory tract with F. tularensis SCHU S4. We show that mice infected with a lethal dose of SCHU S4, via an intra-nasal challenge, could be protected with levofloxacin treatment. This protection was evident even when levofloxacin treatment was delayed 72h post-infection. At early time points after levofloxacin treatment, significant numbers of bacteria could be recovered from the lungs and spleens of mice, which was followed by a dramatic disappearance of bacteria from these tissues. Mice successfully treated with levofloxacin were later shown to be almost completely resistant to re-challenge with SCHU S4 by the intra-nasal route. Serum antibody appeared to play an important role in this immunity. Normal mice, when given sera from animals protected by levofloxacin treatment, were solidly protected from a lethal intra-nasal challenge with SCHU S4. The protective antiserum contained high titers of SCHU S4-specific IgG2a, indicating that a strong Th1 response was induced following levofloxacin treatment. Thus, this study describes a potentially valuable animal model for furthering our understanding of respiratory tularemia and provides suggestive evidence that antibody can protect against respiratory infections with virulent F. tularensis.

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Bacillus anthracis is the Gram-positive, spore-forming etiological agent of anthrax, an affliction studied because of its importance as a potential bioweapon. Although in vitro transcriptional responses of macrophages to either spore or anthrax toxins have been previously reported, little is known regarding the impact of infection on gene expression in host tissues. We infected Swiss-Webster mice intranasally with 5 LD(50) of B. anthracis-virulent Ames spores and observed the global transcriptional profiles of various tissues over a 48 h time period. RNA was extracted from spleen, lung, and heart tissues of infected and control mice and examined by Affymetrix GeneChip analysis. Approximately 580 host genes were significantly over or under expressed among the lung, spleen, and heart tissues at 8 and 48 h time points. Expression of genes encoding for surfactant and major histocompatibility complex (MHC) presentation was diminished during the early phase of infection in lungs. By 48 h, a significant number of genes were modulated in the heart, including up-regulation of calcium-binding-related gene expression, and down-regulation of multiple genes related to cell adhesion, formation of the extracellular matrix, and the cell cytoskeleton. Interestingly, the spleen 8h post-infection showed striking increases in the expression of genes that encode hydrolytic enzymes, and these levels remained elevated throughout infection. Further, genes involving antigen presentation and interferon responses were down-regulated in the spleen at 8 h. In late stages of infection, splenic genes related to the inflammatory response were up-regulated. This study is the first to describe the in vivo global transcriptional response of multiple organs during inhalational anthrax. Although numerous genes related to the host immunological response and certain protection mechanisms were up-regulated in these organs, a vast list of genes important for fully developing and maintaining this response were decreased. Additionally, the lung, spleen, and heart showed differential responses to the infection, further validating the demand for a better understanding of anthrax pathogenesis in order to design therapies against novel targets.

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