A culture-free biphasic approach for sensitive and rapid detection of pathogens in dried whole-blood matrix.

Blood stream infections (BSIs) cause high mortality, and their rapid detection remains a significant diagnostic challenge. Timely and informed administration of antibiotics can significantly improve patient outcomes. However, blood culture, which takes up to 5 d for a negative result, followed by PCR remains the gold standard in diagnosing BSI. Here, we introduce a new approach to blood-based diagnostics where large blood volumes can be rapidly dried, resulting in inactivation of the inhibitory components in blood. Further thermal treatments then generate a physical microscale and nanoscale fluidic network inside the dried matrix to allow access to target nucleic acid. The amplification enzymes and primers initiate the reaction within the dried blood matrix through these networks, precluding any need for conventional nucleic acid purification. High heme background is confined to the solid phase, while amplicons are enriched in the clear supernatant (liquid phase), giving fluorescence change comparable to purified DNA reactions. We demonstrate single-molecule sensitivity using a loop-mediated isothermal amplification reaction in our platform and detect a broad spectrum of pathogens, including gram-positive methicillin-resistant and methicillin-susceptible Staphylococcus aureus bacteria, gram-negative Escherichia coli bacteria, and Candida albicans (fungus) from whole blood with a limit of detection (LOD) of 1.2 colony-forming units (CFU)/mL from 0.8 to 1 mL of starting blood volume. We validated our assay using 63 clinical samples (100% sensitivity and specificity) and significantly reduced sample-to-result time from over 20 h to <2.5 h. The reduction in instrumentation complexity and costs compared to blood culture and alternate molecular diagnostic platforms can have broad applications in healthcare systems in developed world and resource-limited settings.

Efficacy of endotracheal tube suctioning in intubated intensive care unit patients determined by in vivo catheter-based optical coherence tomography-a pilot study.

BACKGROUND: Mechanical ventilation using an endotracheal tube (ETT) is one of the critical interventions given to patients in the intensive care unit (ICU). ETTs are associated with the formation of biofilms, placing patients at increased risk for developing ventilator-associated pneumonia (VAP). ETT suctioning is used to remove secretions, reduce bacterial colonization, and reduce the rate of biofilm formation. However, current standard-of-care suctioning procedures do not adequately eliminate all secretions from the ETT. METHODS: This observational study was conducted in a cohort of 4 subjects admitted to the ICU and intubated with an ETT, irrespective of ethnicity, gender, or race. A total of 23 suctioning procedures were evaluated with in vivo three-dimensional (3D) optical coherence tomography (OCT) imaging, before and after suctioning. A secretion density metric was derived from the OCT data to quantify the amount of secretions present within the ETT, and an attenuation coefficient metric was derived to detect and quantify the presence of biofilms. Analyzed OCT images were correlated with clinical and microscopy data. RESULTS: Data obtained suggests that the current standard-of-care suctioning procedure is inefficient at clearing secretions or preventing the formation of biofilms. The presence of biofilms was corroborated with both post-intubation microscopy of the ETTs, as well as with clinical data. CONCLUSIONS: We conclude that the standard-of-care suctioning method does not eliminate secretions nor reduce the formation of biofilm in ETTs. Our in situ imaging method was sensitive to the presence of secretions, biofilms, and quantitative, and can be used for investigating different suctioning protocols in the future.

Low-Complexity System and Algorithm for an Emergency Ventilator Sensor and Alarm.

In response to anticipated shortages of ventilators caused by the COVID-19 pandemic, many organizations have designed low-cost emergency ventilators. Many of these devices are pressure-cycled pneumatic ventilators, which are easy to produce but often do not include the sensing or alarm features found on commercial ventilators. This work reports a low-cost, easy-to-produce electronic sensor and alarm system for pressure-cycled ventilators that estimates clinically useful metrics such as pressure and respiratory rate and sounds an alarm when the ventilator malfunctions. A low-complexity signal processing algorithm uses a pair of nonlinear recursive envelope trackers to monitor the signal from an electronic pressure sensor connected to the patient airway. The algorithm, inspired by those used in hearing aids, requires little memory and performs only a few calculations on each sample so that it can run on nearly any microcontroller.

In vivo detection of endotracheal tube biofilms in intubated critical care patients using catheter-based optical coherence tomography.

The formation of biofilms in the endotracheal tubes (ETTs) of intubated patients on mechanical ventilation is associated with a greater risk of ventilator-associated pneumonia and death. New technologies are needed to detect and monitor ETTs in vivo for the presence of these biofilms. Longitudinal OCT imaging was performed in mechanically ventilated subjects at 24-hour intervals until extubation to detect the formation and temporal changes of in vivo ETT biofilms. OCT-derived attenuation coefficient images were used to differentiate between mucus and biofilm. Extubated ETTs were examined with optical and electron microscopy, and all imaging results were correlated with standard-of-care clinical test reports. OCT and attenuation coefficient images from four subjects were positive for ETT biofilms and were negative for two subjects. The processed and stained extubated ETTs and clinical reports confirmed the presence/absence of biofilms in all subjects. Our findings confirm that OCT can detect and differentiate between biofilm-positive and biofilm-negative groups (P < 10-5 ). OCT image-based features may serve as biomarkers for direct in vivo detection of ETT biofilms and help drive investigation of new management strategies to reduce the incidence of VAP.