Evaluation of a field deployable, high-throughput RT-LAMP device as an early warning system for COVID-19 through SARS-CoV-2 measurements in wastewater.

Quantification of SARS-CoV-2 RNA copies in wastewater can be used to estimate COVID-19 prevalence in communities. While such results are important for mitigating disease spread, SARS-CoV-2 measurements require sophisticated equipment and trained personnel, for which a centralized laboratory is necessary. This significantly impacts the time to result, defeating its purpose as an early warning detection tool. The objective of this study was to evaluate a field portable device (called MINI) for detecting SARS-CoV-2 viral loads in wastewater using real-time reverse transcriptase loop-mediated isothermal amplification (real-time RT-LAMP). The device was tested using wastewater samples collected from buildings (with 430 to 1430 inhabitants) that had known COVID-19-positive cases. Results show comparable performance of RT-LAMP against reverse transcriptase polymerase chain reaction (RT-qPCR) when detecting SARS-CoV-2 copies in wastewater. Both RT-LAMP and RT-qPCR detected SARS-CoV-2 in wastewater from buildings with at least three positive individuals within a 6-day time frame prior to diagnosis. The large 96-well throughput provided by MINI provided scalability to multi-building detection. The portability of the MINI device enabled decentralized on-site detection, significantly reducing the time to result. The overall findings support the use of RT-LAMP within the MINI configuration as an early detection system for COVID-19 infection using wastewater collected at the building scale.

Mapping of brain stem neuronal circuitry active during swallowing.

A poorly understood neural circuit in the brain stem controls swallowing. This experiment studied the swallowing circuit in the rat brain stem by means of fos immunocytochemistry. The fos protein is a marker of activated neurons, and under experimental conditions, repetition of a behavior causes the fos protein to be produced in the neurons involved in that behavior. The fos technique has been successfully used to delineate neural circuits involved in reflex glottic closure, cough, and vocalization; however, the technique has not been used to map the swallowing circuit. Nine rats were used in this study. Swallows were evoked in anesthetized rats for 1 hour, then, after a 4-hour delay to allow maximum fos production, the rats were painlessly sacrificed by perfusion. The brain stems were removed and sectioned in the frontal plane, and every fourth section was immunoreacted for fos protein. All sections were examined by light microscopy, and cells positive for fos were marked on drawings of brain stem structures for different levels throughout the brain stem. Control animals underwent sham experiments. After subtraction of the areas of fos labeling seen in controls, all experimental rats showed fos-labeled neurons in very discrete and localized areas, including practically all regions implicated by prior neurophysiology studies of swallowing. The distribution of labeled neurons was more dispersed through the brain stem than current theories of swallowing would suggest. Specifically, recent studies of swallowing control have focused on the nucleus of the solitary tract (NST) and the region surrounding the nucleus ambiguus (periambigual area) just rostral to the obex. These areas contained fos-labeled neurons, but unexpectedly, heavy labeling was found in the same areas caudal to the obex. Areas containing the heaviest labeling were specific subnuclei of the NST and surrounding reticular formation; the periambigual area; and the intermediate reticular zone in the pons and caudal medulla. Interestingly, none of these anatomic structures had uniform fos labeling; this finding suggests that the unlabeled areas are involved in other oromotor behaviors, or that the specific protocol did not activate the full population of swallowing-related neurons. A notable finding of this study is a candidate for the central pattern generator (CPG) of swallowing. Careful lesioning studies in cats strongly suggest that a region in the rostral-medial medulla contains the CPG for swallowing, although the exact location of the CPG was never pinpointed. In the homologous region of the rat brain stem, fos labeling was only found in a small group of neurons within the gigantocellular reticular formation that may be a candidate for the CPG. In summary, correlation with prior physiology experiments suggests that this experiment appears to have delineated many, if not all, of the components of the swallowing circuit for the first time in any mammal. In addition, other areas were found that might also be swallowing-related. One notable example is a small group of fos-labeled cells that may be the CPG for swallowing. Further studies are required to clarify the specific roles of the fos-labeled neurons seen in this study.

Safety of esophagoscopy in the irradiated esophagus.

Carcinoma of the esophagus is a disease with a poor prognosis. Surgery is considered the treatment of choice. Patients who are surgically unresectable may receive radiotherapy, plus or minus chemotherapy. While this offers reasonable palliation, a significant number of patients develop benign or malignant strictures. Frequent esophagoscopy with dilation is required if they are to swallow. Patients with strictures or malignancy of the esophagus are more prone to complications if they undergo an esophagoscopy. We examined the morbidity of esophagoscopy in a group of patients who had received radiotherapy for carcinoma of the esophagus. Over a 10-year period, 21 patients receiving irradiation for carcinoma of the esophagus were examined. All patients underwent esophagoscopy for staging and diagnosis. The tumor locations were upper (6), middle (6), and lower (9) esophagus. The T-stages were 1 (7), 2 (6), 3 (6), and 4 (2). Five patients had dilation of a malignant stricture at the time of diagnosis. Fever developed and resolved within 24 hours in 2 patients. Following completion of irradiation, a total of 83 esophagoscopies were performed (range 1 to 11 per patient). Six patients with no tumor or stricture underwent 8 esophagoscopies (3 rigid, 5 flexible). There was 1 episode of minimal bleeding and 1 fever that resolved within 24 hours. Ten patients with malignant strictures underwent 62 esophagoscopies (5 rigid, 57 flexible). There were 17 (27%) episodes of fever (all resolved within 24 hours) and 14 (22%) episodes of bleeding. Five patients with benign strictures underwent 13 esophagoscopies (2 rigid, 11 flexible). One patient had a perforation that resolved with conservative treatment, and 1 patient developed an epidural abscess 2 months following dilation. This patient is the only one that required surgical intervention and had prolonged hospitalization. Esophagoscopy of the irradiated esophagus can be performed relatively safely and excellent palliation obtained. Morbidity consisting of minimal bleeding, and fever lasting less than 24 hours, is frequent but self-limited.