SARS-CoV-2 RNA load and detection rate in exhaled breath condensate collected from COVID-19 patients infected with Delta variant.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Delta variant is considered responsible for worldwide surges in coronavirus disease 2019 (COVID-19) cases in 2021, with increased infectivity compared to the wild type (Wuhan-1). In a previous study, we identified temporal changes in wild-type SARS-CoV-2 RNA load and detection rate in EBC collected from COVID-19 patients. The primary objective of this study was to clarify temporal changes in Delta-variant SARS-CoV-2 RNA load and detection rates in EBC collected from patients, and to validate the feasibility of Delta-variant SARS-CoV-2 RNA detection from EBC for diagnosing COVID-19. The secondary objective was to compare SARS-CoV-2 RNA loads in EBC between Delta-variant and wild-type. Subjects were 41 COVID-19 patients infected with the Delta-variant. EBC samples were collected from subjects on the day of or the day after admission using R-tube® (Respiratory Research, Austin, Texas, USA), as in our previous study. SARS-CoV-2 RNA in EBC samples was detected and quantified by RT-PCR assay targeting the E gene, using the same settings and reagents as in the previous study. The results indicated that SARS-CoV-2 RNA load in EBC collected from subjects infected with Delta-variant decreased exponentially with the passage of days from symptom onset. Sustained high detection rates support the feasibility of Delta-variant SARS-CoV-2 RNA detection from EBC by RT-PCR assay as a diagnostic test for COVID-19 within 8 d of onset. SARS-CoV-2 RNA load in EBC collected 2-8 d from onset was significantly higher in Delta-variant-infected subjects than in wild-type-infected subjects on a day-to-day basis (p= 0.005-0.029). However, because of the heterogeneity of the study cohort, conclusions cannot be reached regarding differences in viral RNA load between strains, regardless of the timing of EBC collection.

RT-PCR diagnosis of COVID-19 from exhaled breath condensate: a clinical study.

Current diagnostic testing for coronavirus disease 2019 (COVID-19) is based on detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in nasopharyngeal swab samples by reverse transcription polymerase chain reaction (RT-PCR). However, this test is associated with increased risks of viral dissemination and environmental contamination and shows relatively low sensitivity, attributable to technical deficiencies in the sampling method. Given that COVID-19 is transmitted via exhaled aerosols and droplets, and that exhaled breath condensate (EBC) is an established modality for sampling exhaled aerosols, detection of SARS-CoV-2 in EBC offers a promising diagnostic approach. However, current knowledge on the detection and load of the virus in EBC collected from COVID-19 patients remains limited and inconsistent. The objective of the study was to quantify the viral load in EBC collected from COVID-19 patients and to validate the feasibility of SARS-CoV-2 detection from EBC as a diagnostic test for the infection. EBC samples were collected from 48 COVID-19 patients using a collection device, and viral loads were quantified by RT-PCR targeting the E gene. Changes in detection rates and viral loads relative to patient characteristics and days since disease onset were statistically evaluated. Need for mechanical ventilation was significantly associated with higher viral load (p< 0.05). Need for oxygen administration or mechanical ventilation, less than 3 d since onset, and presence of cough or fever were significantly associated with higher detection rates (p< 0.05). Among spontaneously breathing patients, viral load in EBC attenuated exponentially over time. The detection rate was 86% at 2 d since onset and deteriorated thereafter. In mechanically ventilated patients, detection rate and viral load were high regardless of days since onset. These results support the feasibility of using RT-PCR to detect SARS-CoV-2 from EBC for COVID-19 patients within 2 d of symptom onset.

A short perspective on a COVID-19 clinical study: 'diagnosis of COVID-19 by RT-PCR using exhale breath condensate samples'.

Diagnosis of SARS-COV-2 infection (COVID-19) is currently based on detection of the viral RNA in nasopharyngeal swab samples by reverse transcription polymerase chain reaction (RT-PCR). However, sampling via nasopharyngeal swabs frequently provokes sneezing or coughing, which results in increased risk of the viral dissemination and environmental contamination. Furthermore, the sensitivity associated with the PCR tests s limited to 60%-70%, which is mainly attributable to technical deficiency in sampling. Given that the disease is transmitted via exhaled aerosol and droplets, and that the exhaled breath condensate (EBC) is the established modality for sampling exhaled aerosol, detection of the viral RNA in EBC is a promising approach for safe and efficient diagnosis of the disease. Subjects are those patients who are diagnosed with COVID-19 by positive nasopharyngeal swab PCR test and admitted to Saitama Medical Center, Japan. EBC samples will be collected using an R-tube® or R-tubeVent® device. Collected EBC samples will be introduced into a nucleic acid purifier. The purified nucleic acids will undergo amplification through RT-PCR for detection and quantification of SARS-COV-2 RNA. To date we have collected eight samples from seven subjects. Among them, two samples from two subjects tested positive for SARS-COV-2 RNA by the RT-PCR. Reflecting the second wave of COVID-19 prevalence in Japan, new admissions of COVID-19 patients to the Saitama Medical Center are increasing, and we are expecting to collect at least 50 EBC samples from 25 patients before the end of this year.

Possible involvement of Notch signaling in the pathogenesis of Buerger's disease.

PURPOSE: Under pathological conditions, the Notch signal pathway is involved in the inflammatory process in arteriosclerosis, atherosclerosis and angiogenesis under ischemic conditions. The purpose of this study was to observe whether or not Buerger's disease is associated with Notch signal activation. METHODS: All the patients were diagnosed between 1980 and 2009 at Nagoya University Hospital. Twenty-two specimens from 12 patients with Buerger's disease (TAO) and 13 specimens from nine patients with arteriosclerosis obliterans (ASO) were analyzed by immunohistochemistry for Notch1, Jagged-1 (a Notch ligand) and Hes-1 (a Notch 1 target transcription factor). RESULTS: Notch1 and Jagged-1 were highly expressed in the endothelium in the new vasa vasorum and in the smooth muscle cells in the media of specimens from both groups. These Notch-related proteins were also remarkably expressed in inflammatory cells in the intima of specimens from TAO patients. Fewer inflammatory cells expressed Notch-related proteins in atheromatous plaques (Notch1 (%): 8.4 ± 0.76 versus 1.3 ± 0.43, P < 0.001; Jagged-1(%): 9.3 ± 1.1 versus 5.2 ± 1.1, P = 0.03). Indeed, Hes-1, which is a transcription factor downstream of Notch1, was remarkably expressed in the endothelium of new capillary vessels and inflammatory cells in TAO patients. Notch1-positive mononuclear cells were also seen in the thrombus in samples from the TAO group. CONCLUSIONS: Our findings are the first demonstration that Notch signal activation in inflammatory cells may be involved in the pathophysiological mechanism underlying Buerger's disease.