Browsing by Subject "Sepsis"

Background The diagnosis of sepsis is challenging because the infection can be caused by more than 50 species of pathogens that might exist in the bloodstream in very low concentrations, e.g., less than 1 colony-forming unit/ml. As a result, among the current sepsis diagnostic methods there is an unsatisfactory trade-off between the assay time and the specificity of the derived diagnostic information. Although the present qPCR-based test is more specific than biomarker detection and faster than culturing, its 6 ~ 10 hr turnaround remains suboptimal relative to the 7.6%/hr rapid deterioration of the survival rate, and the 3 hr hands-on time is labor-intensive. To address these issues, this work aims to utilize the advances in microfluidic technologies to expedite and automate the ``nucleic acid purification - qPCR sequence detection'' workflow.

Methods and Results This task is evaluated to be best approached by combining immiscible phase filtration (IPF) and digital microfluidic droplet actuation (DM) on a fluidic device. In IPF, as nucleic acid-bound magnetic beads are transported from an aqueous phase to an immiscible phase, the carryover of aqueous contaminants is minimized by the high interfacial tension. Thus, unlike a conventional bead-based assay, the necessary degree of purification can be attained in a few wash steps. After IPF reduces the sample volume from a milliliter-sized lysate to a microliter-sized eluent, DM can be used to automatically prepare the PCR mixture. This begins with compartmenting the eluent in accordance with the desired number of multiplex qPCR reactions, and then transporting droplets of the PCR reagents to mix with the eluent droplets. Under the outlined approach, the IPF - DM integration should lead to a notably reduced turnaround and a hands-free ``lysate-to-answer'' operation.

As the first step towards such a diagnostic device, the primary objective of this thesis is to verify the feasibility of the IPF - DM integration. This is achieved in four phases. First, the suitable assays, fluidic device, and auxiliary systems are developed. Second, the extent of purification obtained per IPF wash, and hence the number of washes needed for uninhibited qPCR, are estimated via off-chip UV absorbance measurement and on-chip qPCR. Third, the performance of on-chip qPCR, particularly the copy number - threshold cycle correlation, is characterized. Lastly, the above developments accumulate to an experiment that includes the following on-chip steps: DNA purification by IPF, PCR mixture preparation via DM, and target quantification using qPCR - thereby demonstrating the core procedures in the proposed approach.

Conclusions It is proposed to expedite and automate qPCR-based multiplex sparse pathogen detection by combining IPF and DM on a fluidic device. As a start, this work demonstrated the feasibility of the IPF - DM integration. However, a more thermally robust device structure will be needed for later quantitative investigations, e.g., improving the bead - buffer mixing. Importantly, evidences indicate that future iterations of the IPF - DM fluidic device could reduce the sample-to-answer time by 75% to 1.5 hr and decrease the hands-on time by 90% to approximately 20 min.

Nitric oxide (NO) is an endogenous gaseous signaling molecule produced by three NO synthase isoforms (NOS1, 2, 3) and important in host defense. The induction of NOS2 during bacterial sepsis is critical for pathogen clearance but its sustained activation has long been associated with increased mortality secondary to multiple organ dysfunction syndrome (MODS). High levels of NO produced by NOS2 incite intrinsic cellular dysfunction, in part by damaging macromolecules through nitration and/or nitrosylation. These include mitochondrial DNA (mtDNA) and enzymes of key mitochondrial pathways required for maintenance of normal O2 utilization and energy homeostasis. However, animal studies and clinical trials inhibiting NOS2 have demonstrated pronounced organ dysfunction and increased mortality in response to live bacterial infections, confirming that NOS2 confers pro-survival benefits. Of particular interest here, the constitutive NOS1 and NOS3 have been linked to the up-regulation of nuclear genes involved in mitochondrial biogenesis but no comparable role has been described for NOS2. Therefore, I hypothesized that NOS2 is indispensible for host protection but must be tightly regulated to ensure NO levels are high enough to activate mitochondrial and other pro-survival genes, but below the threshold for cellular damage.

This hypothesis was explored with two major Aims. The first Aim was to define the role of NOS2 in the activation of mitochondrial biogenesis in the heart of E. coli-treated mice. The second was to investigate the ability of NOS2 to be transcriptionally regulated by an enzyme previously shown to induce mitochondrial biogenesis, heme oxygenase-1 (HO-1). This hypothesis was tested using an in vivo model of sublethal heat-killed E. coli (HkEC) peritonitis in C57B/L6 (Wt), NOS2-/-, and TLR4-/- mice. Additionally, in vitro systems of mouse AML-12 or Hepa 1-6 cells pretreated with HO-1 activators or Hmox1 shRNA prior to inflammatory challenge with lipopolysaccharide (LPS) +/- tumor necrosis factor-α (TNF-α). For the first Aim, Wt, NOS2-/-, and TLR4-/- mice were treated with (HkEC and cardiac tissue analyzed for mitochondrial function, expression of nuclear and mitochondrial proteins needed for mitochondrial biogenesis, and histological expression of NOS2 and TLR4 relative to changes in mitochondrial mass. For the second Aim, Wt mice were pretreated with hemin or carbon monoxide (CO) to activate HO-1 prior to HkEC-peritonitis. Liver tissue in these animals was evaluated at four hours for HO-1 induction, Nos2 mRNA expression, cytokine profiles, and nuclear factor (NF)-κB activation. Liver cell lines were pretreated with hemin, CO-releasing molecule (CORM), or bilirubin one hour before LPS exposure and the Nos2 transcriptional response evaluated at two and 24 hours. The MTT assay was used to confirm that in vitro treatments were not lethal.

These studies demonstrated that HkEC induced mtDNA damage in the heart that was repaired in Wt mice but not in NOS2-deficient mice. In KO mice, sustained mtDNA damage was associated with the reduced expression of nuclear (NRF-1, PGC-1α) and mitochondrial (Tfam, Pol-γ) proteins needed for mitochondrial biogenesis. The findings thus supported that NOS2 is required for mitochondrial biogenesis in the heart during Gram-negative challenge. Evaluation of the relationship between HO-1 and NOS2 in murine liver was more complex; HO-1 activation in HkEC-treated Wt mice attenuated 4-hour Nos2 gene transcription. In liver cell lines, hemin, CORM, and bilirubin were unable to suppress Nos2 expression at the time of maximal induction (2 hours). Nos2 was, however, suppressed by 24 hours, suggesting that the regulatory impact of HO-1 induction was not engaged early enough to reduce Nos2 transcription at 2 hours. It is concluded that NOS2 induction in bacterial sepsis optimizes the expression of the mitochondrial biogenesis transcriptional program, which subsequently can also be regulated by HO-1/CO in murine liver. This provides a potential new mechanism by which immune suppression and mitochondrial repair can occur in tandem during the acute inflammatory response.

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In vivo imaging is an important scientific tool for studying bio-molecular interactions, but lack of preserved functionality during imaging restricts scientists’ abilities to gain critical knowledge. Structure can be preserved while using high-resolution optical imaging by utilizing window chambers in murine models1, yet the use of anesthesia for immobilization is problematic. Anesthesia affects tissue oxygenation2, blood cell velocities3, immunosuppression4, and allowable duration of imaging5–thus its usage restricts in vivo bio-molecular imaging accuracy and duration.

Developing a portable imaging system that attaches to murine dorsal window chambers enables imaging without anesthesia, avoiding previous drawbacks of window chamber models. A raspberry pi camera (RPI-CAM-V2, Raspberry Pi) was modified for microscopy and used alongside 3D printed panels for attaching the camera, optical filters, and LED light source to murine window chambers. Multiple applications for the portable system were developed, each requiring their own setup of filters and stimulating LEDs. The system is powered by a Raspberry Pi 3 Model B single-board computer (RASPBERRYPI3-MODB-1GB, Raspberry Pi), allowing for stream-lined data acquisition.

Imaging tissue oxygenation was the first application developed for the portable system. Oxygen sensing boron nanoparticles were injected into window chambers, while a UV LED was used to stimulate fluorescent and phosphorescent signals. When stimulated by UV light, the boron nanoparticles emit fluorescence and phosphorescence. Fluorescence is stable regardless of oxygenation, while phosphorescence signal from the nanoparticles is quenched in the presence of higher oxygenations. The ratio of fluorescence to phosphorescence was used to calculate oxygen concentration maps of window chamber tissue. Tissue oxygenations in awake and anesthetized mice inhaling varied oxygen concentrations were analyzed. In 5 awake nude mice inhaling 20% O2, the median partial pressure of oxygen was measured as 49 mmHg within their window chambers. From a one-tailed t-test with a false positive correction, 3 of the mice had significantly higher (p ≤ 0.05) tissue oxygenation while anesthetized compared to the awake measurements.

Developing the portable systems ability to image blood cells was another focus of this project. Blood cells were visible with white LED exposure. A frame rate of 30 frames/second was adequate for tracking cell motion while allowing for the highest resolution possible with the system. Blood cell velocities in a mouse awake and anesthetized were analyzed, while also observing change in blood cell velocities during sepsis that was induced by cecal ligation puncture (CLP). Three days after CLP, the mean awake blood cell velocity was measured as 0.21 ± 0.03 mm/s, while the mean anesthetized blood cell velocity was measured as 0.080 ± 0.002 mm/s. Six days after CLP, the awake measurement had reduced to 0.019 ± 0.005 mm/s, while the anesthetized measurement was reduced to 0.031 ± 0.002 mm/s (91% decrease in awake measurement, 61% decrease in anesthetized measurement). A two-way ANOVA on the factors of anesthesia and time post-CLP performed on multiple vessel regions calculated significance (p ≤ 0.05) for both of these factors on blood cell velocities within the pilot mouse’s window chamber.

Noting the differences between data collected on awake and anesthetized mice, our system has been validated as a tool for real-time imaging of tissue without the observed effects of anesthesia. By avoiding anesthesia, the developed device allows for continual data acquisition to increase from hours to days. The system is generalizable, and while only two applications are presented in this study, the system could be modified for imaging fluorescently labeled cells/proteins for other bio-molecular interactions.

Background: Post-cesarean section peritonitis is the leading cause of maternal morbidity and mortality at the main referral hospital in Rwanda. Published data on the management of post-cesarean section peritonitis is limited. This study examined predictors of maternal morbidity and mortality for post-cesarean peritonitis.

Methods: We performed a prospective observational cohort study at the University Teaching Hospital Kigali (CHUK) from January 1 until December 31 2015, followed by a retrospective chart review of all subjects with post-cesarean section peritonitis admitted to CHUK from January 1 until December 31, 2014. All patients admitted with the diagnosis of post-cesarean section peritonitis undergoing exploratory laparotomy at CHUK were enrolled. Patients were followed to either discharge or death. Study variables included baseline demographic/clinical characteristics, admission physical exam, intraoperative findings, and management. Data were analyzed using STATA version 14.

Results: Of the 167 patients enrolled, 81 survived without requiring hysterectomy (49%), 49 survived requiring hysterectomy (29%), and 36 died (22%). In the multivariate analysis, severe sepsis was the most significant predictor of mortality (RR=4.0 [2.2-7.7]) and uterine necrosis was the most significant predictor of hysterectomy (RR=6.3 [1.6-25.2]). There were high rates of antimicrobial resistance (AMR) among the bacterial isolates cultured from intra-abdominal pus, with 52% of bacteria resistant to third-generation cephalosporins.

Conclusions: Post-cesarean section peritonitis carries a high mortality rate in Rwanda. It is also associated with a high rate of hysterectomy. Understanding the disease process and identifying factors associated with outcomes can help guide management during admission.

We present a scalable end-to-end classifier that uses streaming physiological and medication data to accurately predict the onset of sepsis, a life-threatening complication from infections that has high mortality and morbidity. Our proposed framework models the multivariate trajectories of continuous-valued physiological time series using multitask Gaussian processes, seamlessly accounting for the high uncertainty, frequent missingness, and irregular sampling rates typically associated with real clinical data. The Gaussian process is directly connected to a black-box classifier that predicts whether a patient encounter will become septic, chosen in our case to be a recurrent neural network to account for the extreme variability in the length of patient encounters. We show how several approximations scale the computations associated with the Gaussian process in a manner so that the entire system can be trained discriminatively end-to-end using backpropagation. In a large cohort of heterogeneous inpatient encounters at our university health system we find that it outperforms several baselines at predicting sepsis, and yields 33\% and 195\% improved areas under the Receiver Operating Characteristic and Precision Recall curves as compared to the NEWS score currently in use on our own hospital wards.

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Background: Sepsis is a leading cause of death and disability globally. Despite a high burden of sepsis in sub-Saharan Africa, clinical data for sepsis in that setting are limited. We sought to describe the clinical characteristics, management, and outcomes in a cohort of adults and adolescents with sepsis in northern Tanzania. We also assessed for associations between clinical factors and in-hospital mortality.

Methods: We carried out a prospective observational cohort study at Kilimanjaro Christian Medical Centre in Moshi, Tanzania. We collected data on demographics, baseline clinical characteristics, and management, with an emphasis on hours 0-6 after arrival to the Emergency Department. Log risk regression was carried out to assess for associations between demographic and clinical factors and our primary outcome of in-hospital death. Separate multivariable regression analyses were conducted for both antimicrobial administration by hour 6 and administration of intravenous (IV) fluids >1L by hour 6 and the outcome of in-hospital mortality.

Results: Fifty-eight participants were included in our analysis. Seventeen (29.3%) participants died in-hospital. Baseline characteristics associated with inpatient mortality included inability to drink unassisted, respiratory rate >30 breaths per minute, hypoxia, and altered mentation. Less than half of participants received any antimicrobial by hour 6, and most participants received <1L of IV fluids. HIV antibody testing was performed for only one participant in the first 6 hours. On multivariable analysis, neither antimicrobial administration nor IV fluids >1L by hour 6 was associated with inpatient mortality.

Conclusion: Sepsis in northern Tanzania carries a high risk of in-hospital mortality. Further research is urgently needed to establish the highest-yield interventions suited to the unique characteristics of sepsis in sSA.