Background Recently developed rapid real-time reverse transcription PCR (RT-PCR) systems adopting microfluidic thermal cycling technology are ideal for point-of-care (POC) testing for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Because the RNA extraction step before real-time RT-PCR is rate-limiting, a direct RNA extraction method (direct method) that adopts chemical viral lysis and eliminates RNA purification steps is preferable for rapid real-time RT-PCR. In the direct method, selecting the transport medium is essential because it may be introduced into subsequent real-time RT-PCR steps, but might inhibit PCR. However, the influence of transport medium on the combination of the direct method and rapid real-time RT-PCR has been yet unstudied. In the present study, we examined the influence of various transport mediums when combining the direct method and rapid real-time RT-PCR of GeneSoC® (GeneSoC® RT-PCR), the recently developed compact PCR system that adapts novel microfluidic thermal cycling technology.Methods To explore the influence of the transport medium on the GeneSoC® RT-PCR, the concordance of the RNA extraction and direct method was evaluated in the clinical samples collected in viral transport medium (VTM) or eSwab®. The sensitivity of GeneSoC® RT-PCR combined with the direct method was assessed using spiked samples in generic (H2O and PBS) or commercially available transport media (VTM and eSwab®). Analytical sensitivity was examined using clinical specimens collected from the VTM and eSwab®. The inhibitory effect of PCR inhibitors on clinical specimens was assessed using clinical samples diluted 1,000 times.Results While only 1 copy/reaction of RNA was detected in H2O and eSwab® of the spiked samples, a minimum of 5 copies/reaction was detected in PBS (-) and VTM. Among the clinical specimens tested using the direct method, the detection of viral RNA was unstable in the samples containing less than 100 copies/reaction viral RNA in VTM, whereas less than 10 copies/reaction viral RNA were detected in eSwab®. The positive, negative, and overall concordance between the RNA extraction and the direct method was 84%, 100%, and 85%, respectively, in eSwab® samples, whereas the values were 35%, 100%, and 38%, respectively, in VTM samples. When the clinical samples were diluted 1,000 times, GeneSoC® RT-PCR could detect as low as 1.15 copies/reaction RNA using direct method, and the sensitivity was comparable to that of RNA extraction.Conclusion The combination of the direct method and microfluidic rapid PCR machine GeneSoC® has a high sensitivity for detecting SARS-CoV-2 RNA in clinical samples with eSwab® transport medium.

Persistent SARS-CoV-2 infections involve prolonged viral replication and immune system interactions, potentially driving viral evolution and immune escape. This study examines viral characteristics and host gene expression changes in persistent infections. The nasopharyngeal samples from four patients with persistent SARS-CoV-2 infections at Tottori University Hospital, Japan, were analyzed. Viral isolates were cultured, and infectivity was assessed using TCID₅₀ assays. To investigate host responses, RNA sequencing (RNA-seq) was performed to identify differentially expressed genes (DEGs), and Gene Ontology (GO) enrichment analysis mapped affected biological pathways. Viral genome sequencing detected mutations associated with prolonged infection. The results showed significant infectivity differences between early- and late-phase infection. Gene expression analysis revealed a strong early phase of pro-inflammatory response (IL6, TNF, IL1B, CXCL10) followed by immune suppression. GO enrichment analysis highlighted inflammation and cytokine-mediated immune pathways. Genomic sequencing identified mutations in ORF1ab and the spike (S) protein, potentially aiding immune escape. The findings underscore that SARS-CoV-2 adapts during persistent infections, altering infectivity and immune responses. These highlight the need for continued monitoring of prolonged infections to mitigate immune escape and viral evolution.

Background/Objectives: Continuous positive airway pressure (CPAP) is used to treat patients with obstructive sleep apnea (OSA) and has proven clinical efficacy for this condition. However, the objective method to determine the appropriate CPAP level for treatment is still unclear. Patients with OSA typically exhibit irregular respiratory efforts due to obstruction or narrowing of the upper airway during sleep. Treatment with an adequate CPAP level alleviates airway obstruction or narrowing, leading to more regular respiratory patterns. We aimed to develop an objective CPAP titration method using the respiratory regularity index (RRI) derived from respiratory movements. Methods: We conducted daytime CPAP titration in 10 patients with OSA. Respiratory movements were recorded by inductance plethysmography in three conditions: wake, apnea, and during CPAP therapy. The RRI of respiratory movements was calculated in each condition, and the CPAP level with the lowest RRI was identified as the appropriate air pressure level in each patient. CPAP therapy at the appropriate level determined by the study method was conducted for 2 months, after which efficacy was assessed by night polysomnography in the hospital. Results: The fixed air pressure level for the CPAP device was determined as 7.8 ± 0.7 cmH₂O based on our daytime CPAP titration method using the RRI. The apnea–hypopnea index improved significantly from before CPAP therapy (43.1 ± 15.3 h⁻¹) to during CPAP therapy (3.0 ± 2.1 h⁻¹) (p < 0.006). The Epworth Sleepiness Scale score decreased significantly during CPAP therapy. The lowest percutaneous arterial oxygen saturation values in each sleep stage also improved significantly with the CPAP determined by our daytime CPAP titration method. Conclusions: The study findings showed that our daytime CPAP titration method based on the RRI is an effective method for determining individualized appropriate CPAP levels in patients with OSA. The RRI-based daytime CPAP titration method offers a simplified approach to determining the adequate CPAP level for patients with OSA.