Multiplexed detection of SARS-CoV-2 genomic and subgenomic RNA using in situ hybridization.

The widespread Coronavirus Disease 2019 (COVID-19) is caused by infection with the novel coronavirus SARS-CoV-2. Currently, we have a limited toolset available for visualizing SARS-CoV-2 in cells and tissues, particularly in tissues from patients who died from COVID-19. Generally, single-molecule RNA FISH techniques have shown mixed results in formalin fixed paraffin embedded tissues such as those preserved from human autopsies. Here, we present a platform for preparing autopsy tissue for visualizing SARS-CoV-2 RNA using RNA FISH with amplification by hybridization chain reaction (HCR). We developed probe sets that target different regions of SARS-CoV-2 (including ORF1a and N) as well as probe sets that specifically target SARS-CoV-2 subgenomic mRNAs. We validated these probe sets in cell culture and tissues (lung, lymph node, and placenta) from infected patients. Using this technology, we observe distinct subcellular localization patterns of the ORF1a and N regions, with the ORF1a concentrated around the nucleus and the N showing a diffuse distribution across the cytoplasm. In human lung tissue, we performed multiplexed RNA FISH HCR for SARS-CoV-2 and cell-type specific marker genes. We found viral RNA in cells containing the alveolar type 2 (AT2) cell marker gene (SFTPC) and the alveolar macrophage marker gene (MARCO), but did not identify viral RNA in cells containing the alveolar type 1 (AT1) cell marker gene (AGER). Moreover, we observed distinct subcellular localization patterns of viral RNA in AT2 cells and alveolar macrophages, consistent with phagocytosis of infected cells. In sum, we demonstrate the use of RNA FISH HCR for visualizing different RNA species from SARS-CoV-2 in cell lines and FFPE autopsy specimens. Furthermore, we multiplex this assay with probes for cellular genes to determine what cell-types are infected within the lung. We anticipate that this platform could be broadly useful for studying SARS-CoV-2 pathology in tissues as well as extended for other applications including investigating the viral life cycle, viral diagnostics, and drug screening.

Validation of a flash glucose monitoring system in outpatient diabetic cats.

BACKGROUND: Interstitial glucose (IG) concentration measurement using a flash glucose monitoring system (FGMS) is a noninvasive, affordable, and informative method to regulate patients with diabetes mellitus (DM) but has not been fully validated in outpatient cats with DM. OBJECTIVES: To further validate the FreeStyle Libre FGMS in outpatient diabetic cats. ANIMALS: Eight client-owned cats with DM. METHODS: Prospective observational validation study. Tissue glue was used to attach the sensor to the cat. Lin's concordance correlation coefficient (ρc ) was used to compare IG concentrations measured by the FGMS to blood glucose concentrations measured using an automated biochemistry analyzer (ABA) and point-of-care glucometer (POCG). RESULTS: Data from 15 sensor placements in 8 cats were analyzed. Paired IG and ABA glucose concentrations (139 samples) had excellent correlation (ρc  = 0.96) as did IG and POCG glucose concentrations (142 samples, ρc  = 0.92). Sensor failure or displacement were recorded for 12/15 (80%) sensor placements. Median time of sensor activity was 7 days (range, 2-13 days). CONCLUSIONS AND CLINICAL IMPORTANCE: In outpatient cats with DM, the FGMS-measured IG concentration correlated well with ABA-measured blood glucose concentration, but a high rate of sensor failures was observed.

Assessment of postprandial hyperglycemia and circadian fluctuation of glucose concentrations in diabetic dogs using a flash glucose monitoring system.

BACKGROUND: Postprandial hyperglycemia (PPH) and circadian glucose concentration fluctuations recorded in the home environment of dogs with naturally occurring diabetes mellitus (DM) have not been reported. OBJECTIVES: To determine if a flash glucose monitoring system (FGMS; FreeStyle Libre) can detect PPH and circadian fluctuations in glucose concentrations in dogs with variably controlled DM. ANIMALS: Fourteen client-owned dogs with DM. METHODS: Prospective observational study. Interstitial glucose (IG) concentrations measured by the FGMS during a 13-day study period were analyzed. RESULTS: A total of 17, 446 FGMS IG concentrations were analyzed. For all dogs analyzed together, median IG concentration measured within 30 (288 mg/dL), 60 (286 mg/dL), 90 (285 mg/dL), and 120 (285 mg/dL) minutes of meals was each significantly higher than the median IG concentration at all other times (260 mg/dL, 259 mg/dL, 258 mg/dL, and 257 mg/dL, respectively; range, 40-500 mg/dL; P < .001 for each). Median night-time IG concentration measured from all dogs on 3,547 samples recorded between 1:00 am and 6:00 am (268 mg/dL; range, 40-500 mg/dL) was significantly higher than median IG measured on 13, 899 samples at all other time points (259 mg/dL; range, 40-500 mg/dL; P < .001). CONCLUSIONS AND CLINICAL IMPORTANCE: The FGMS can be used for future studies of PPH and circadian fluctuations of glucose concentrations in dogs with DM in their home environment.

Subcellular Detection of SARS-CoV-2 RNA in Human Tissue Reveals Distinct Localization in Alveolar Type 2 Pneumocytes and Alveolar Macrophages.

The widespread coronavirus disease 2019 (COVID-19) is caused by infection with the novel coronavirus SARS-CoV-2. Currently, we have limited understanding of which cells become infected with SARS-CoV-2 in human tissues and where viral RNA localizes on the subcellular level. Here, we present a platform for preparing autopsy tissue for visualizing SARS-CoV-2 RNA using RNA fluorescence in situ hybridization (FISH) with amplification by hybridization chain reaction. We developed probe sets that target different regions of SARS-CoV-2 (including ORF1a and N), as well as probe sets that specifically target SARS-CoV-2 subgenomic mRNAs. We validated these probe sets in cell culture and tissues (lung, lymph node, and placenta) from infected patients. Using this technology, we observe distinct subcellular localization patterns of the ORF1a and N regions. In human lung tissue, we performed multiplexed RNA FISH HCR for SARS-CoV-2 and cell-type-specific marker genes. We found viral RNA in cells containing the alveolar type 2 (AT2) cell marker gene (SFTPC) and the alveolar macrophage marker gene (MARCO) but did not identify viral RNA in cells containing the alveolar type 1 (AT1) cell marker gene (AGER). Moreover, we observed distinct subcellular localization patterns of viral RNA in AT2 cells and alveolar macrophages. In sum, we demonstrate the use of RNA FISH HCR for visualizing different RNA species from SARS-CoV-2 in cell lines and FFPE (formalin fixation and paraffin embedding) autopsy specimens. We anticipate that this platform could be broadly useful for studying SARS-CoV-2 pathology in tissues, as well as extended for other applications, including investigating the viral life cycle, viral diagnostics, and drug screening. IMPORTANCE Here, we developed an in situ RNA detection assay for RNA generated by the SARS-CoV-2 virus. We found viral RNA in lung, lymph node, and placenta samples from pathology specimens from COVID patients. Using high-magnification microscopy, we can visualize the subcellular distribution of these RNA in single cells.

Vesicovaginal fistula in a dog with urinary incontinence.

CASE DESCRIPTION: A 5-year-old spayed female Maltese mixed-breed dog was referred for evaluation because of severe urinary incontinence refractory to medical management. CLINICAL FINDINGS: Physical examination revealed constant dribbling of urine and urine scalding. Culture of a urine sample yielded methicillin-resistant Staphylococcus pseudintermedius and Proteus mirabilis. Abdominal ultrasonographic examination revealed absence of the left kidney, a small, nondistended urinary bladder, and diffuse hepatopathy. Urinary incontinence persisted despite appropriate antimicrobial treatment. Cystourethroscopy and vaginoscopy were subsequently performed and revealed a hypoplastic bladder and a vesicovaginal fistula with urinary leakage through the vaginal diverticulum; no left ureterovesicular junction was identified, consistent with suspected left renal aplasia. TREATMENT AND OUTCOME: Exploratory laparotomy was performed, and the cranial aspect of the vagina was circumferentially ligated immediately caudal to the fistula. The urinary incontinence resolved immediately after surgery, and lower urinary tract signs improved over the next 2 weeks. Moderate urinary incontinence recurred approximately 6 months later, and a urinary tract infection with Escherichia coli was subsequently identified and treated; clinical signs resolved ≤ 48 hours after treatment was initiated. CLINICAL RELEVANCE: To the author's knowledge, vesicovaginal fistulas in dogs have not been previously described and should be considered a differential diagnosis for persistent urinary incontinence and recurrent urinary tract infections in female dogs. Vaginoscopy in addition to cystourethroscopy was required to identify the abnormality in this patient. Because multiple concurrent anomalies can be present, both procedures should be performed in female dogs with these clinical signs, even if an abnormality is identified cystoscopically.

Survival analysis of hypotensive cats admitted to an intensive care unit with or without hyperlactatemia: 39 cases (2005-2011).

OBJECTIVE To examine the association between blood lactate concentration and survival to hospital discharge in critically ill hypotensive cats. DESIGN Retrospective case series. ANIMALS 39 cats admitted to an intensive care unit of a university veterinary hospital between January 2005 and December 2011 for which blood lactate concentration was recorded ≤ 1 hour before or after a Doppler-derived arterial blood pressure measurement ≤ 90 mm Hg (ie, hypotension) was obtained. PROCEDURES Medical records of each cat were reviewed to assess survival to hospital discharge, illness severity, duration of hospitalization, age, body weight, and PCV. Results were compared between hypotensive cats with and without hyperlactatemia (blood lactate concentration ≥ 2.5 mmol/L). RESULTS 6 of 39 (15%) hypotensive cats survived to hospital discharge. Twelve (31%) cats were normolactatemic (blood lactate concentration < 2.5 mmol/L), and 27 (69%) were hyperlactatemic. Hypotensive cats with normolactatemia had a higher blood pressure and higher survival rate than hypotensive cats with hyperlactatemia. Five-day Kaplan-Meier survival rates were 57% for normolactatemic cats and 17% for hyperlactatemic cats. Age, body weight, duration of hospitalization, PCV, and illness severity did not differ significantly between hypotensive cats with and without hyperlactatemia. CONCLUSIONS AND CLINICAL RELEVANCE Hypotensive, normolactatemic cats in an intensive care unit had a significantly greater chance of survival to hospital discharge than their hyperlactatemic counterparts. Blood lactate concentration may be a useful prognostic indicator for this patient population when used in conjunction with other clinical and laboratory findings.