Does the timing of saliva collection affect the diagnosis of SARS-CoV-2 infection?

We evaluated the optimal timing of saliva sample collection to diagnose the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. We obtained 150 saliva samples at four specific time points from 13 patients with confirmed SARS-CoV-2 infection. The time points were (1) early morning (immediately after waking), (2) immediately after breakfast before tooth brushing, (3) 2 h after breakfast, and (4) before lunch. On the 2nd hospital day, patients collected saliva at the four time points by themselves. We collected samples at two time points, (1) and (3), from the 3rd hospital day to day 9 following symptom onset. In 52 samples collected at the four time points, there was no significant difference. Meanwhile, there was no significant difference in the positive proportion or the viral load between the two time points in both analyses by the day from symptom onset and by all samples. In this study, there was no difference in the positive proportions in saliva collected at various time points within 9 days after symptom onset. The timing of saliva collection was not affected by the diagnosis of SARS-CoV-2 infection.

A homogeneous assay to determine high-density lipoprotein subclass cholesterol in serum.

Existing methods to measure high-density lipoprotein cholesterol (HDL-C) subclasses (HDL2-C and HDL3-C) are complex and require proficiency, and thus there is a need for a convenient, homogeneous assay to determine HDL-C subclasses in serum. Here, cholesterol reactivities in lipoprotein fractions [HDL2, HDL3, low-density lipoprotein (LDL), and very-low-density lipoprotein (VLDL)] toward polyethylene glycol (PEG)-modified enzymes were determined in the presence of varying concentrations of dextran sulfate and magnesium nitrate. Particle sizes formed in the lipoprotein fractions were measured by dynamic light scattering. We optimized the concentrations of dextran sulfate and magnesium nitrate before assay with PEG-modified enzymes to provide selectivity for HDL3-C. On addition of dextran sulfate and magnesium nitrate, the sizes of particles of HDL2, LDL, and VLDL increased, but the size of HDL3 fraction particles remained constant, allowing only HDL3-C to participate in coupled reactions with the PEG-modified enzymes. In serum from both healthy volunteers and patients with type 2 diabetes, a good correlation was observed between the proposed assay and ultracentrifugation in the determination of HDL-C subclasses. The assay proposed here enables convenient and accurate determination of HDL-C subclasses in serum on a general automatic analyzer and enables low-cost routine diagnosis without preprocessing.

Evaluation of false positives in the SARS-CoV-2 quantitative antigen test.

INTRODUCTION: Highly sensitive reagents for detecting SARS-CoV-2 antigens have been developed for accurate and rapid diagnosis till date. In this study, we aim to clarify the frequency of false-positive reactions and reveal their details in SARS-CoV-2 quantitative antigen test using an automated laboratory device. METHODS: Nasopharyngeal swab samples (n = 4992) and saliva samples (n = 5430) were collected. We measured their SARS-CoV-2 antigen using Lumipulse® Presto SARS-CoV-2 Ag and performed a nucleic acid amplification test (NAAT) using the Ampdirect™ 2019 Novel Coronavirus Detection Kit as needed. The results obtained from each detection test were compared accordingly. RESULTS: There were 304 nasopharyngeal samples and 114 saliva samples were positive in the Lumipulse® Presto SARS-CoV-2 Ag test. All positive nasopharyngeal samples in the antigen test were also positive for NAAT. In contrast, only three (2.6%) of all the positive saliva samples in the antigen test were negative for NAAT. One showed no linearity with a dilute solution in the dilution test. Additionally, the quantitative antigen levels of all the three samples did not decrease after reaction with the anti-SARS-CoV-2 antibody. CONCLUSIONS: The judgment difference between the quantitative antigen test and NAAT seemed to be caused by non-specific reactions in the antigen test. Although the high positive and negative predictive value of this quantitative antigen test could be confirmed, we should consider the possibility of false-positives caused by non-specific reactions and understand the characteristics of antigen testing. We recommend that repeating centrifugation before measurement, especially in saliva samples, should be performed appropriately.

Cationic liposome-mediated delivery of reovirus enhances the tumor cell-killing efficiencies of reovirus in reovirus-resistant tumor cells.

Reovirus induces tumor cell death efficiently and specifically, and thus is currently undergoing clinical testing as an anticancer agent. In the intracellular trafficking of reovirus, proteolytic disassembly of reovirus capsid-proteins and subsequent penetration of viral particles into the cytosol are crucial steps. Cathepsins B and L are largely responsible for the proteolytic disassembly of reovirus. Reovirus efficiently lyses tumor cells exhibiting relatively high activities of cathepsins B and L, while tumor cells with low activities of cathepsins B and L are often refractory to reovirus, probably due to inefficient endo/lysosomal escape. In this study, in order to enhance the tumor cell-killing efficiencies of reovirus by promoting endo/lysosomal escape, especially in reovirus-resistant tumor cells, reovirus was complexed with a cationic liposome transfection reagent. Reovirus alone and reovirus-cationic liposome complex (reoplex) exhibited similar levels of tumor cell-killing efficiencies in reovirus-susceptible tumor cells, while reoplex mediated more than 30% higher levels of tumor cell-killing activities in reovirus-resistant tumor cells than reovirus alone. Reoplex-mediated tumor cell death was efficiently induced in the tumor cells pretreated with cathepsin inhibitors. The mRNA levels of interferon (IFN)-ß and apoptotic genes were significantly elevated following reoplex treatment. These results suggest that cationic liposomes efficiently promoted delivery of reovirus to the cytosol, leading to induction of apoptosis.

Immunogenic properties of Landrace pigs selected for resistance to mycoplasma pneumonia of swine.

Mycoplasma pneumonia of swine (MPS) lung lesions and immunogenic properties were compared between a Landrace line that was genetically selected for reduced incidence of pulmonary MPS lesions, and a non-selected Landrace line. The MPS-selected Landrace line showed significantly lower degrees of pulmonary MPS lesions compared with the non-selected Landrace line. When changes in immunity before and after vaccination were compared, the percentage of B cells in the peripheral blood of the MPS-selected Landrace line was significantly lower than that of the non-selected line. Furthermore, the concentration of growth hormone and the mitogen activity of peripheral blood mononuclear cells in the MPS-selected Landrace line showed significantly (P < 0.05) lower increases after vaccination than the non-selected line. Conversely, the concentration of peripheral blood interferon (IFN)-γ and salivary immunoglobulin A (IgA) after Mycoplasma hyopneumoniae vaccination was significantly higher in the MPS-selected Landrace line than in the non-selected line. Gene expression of toll-like receptor (TLR)2 and TLR4 was significantly higher in the MPS-selected Landrace line in immune tissues, with the exception of the hilar lymph nodes. The present results suggest that peripheral blood IFN-γ, salivary IgA TLR2, and TLR4 are important immunological factors influencing the development of MPS lesions.