Establishment of an in-house real-time RT-PCR assay for the detection of severe acute respiratory syndrome coronavirus 2 using the first World Health Organization international standard in a resource-limited country.

BACKGROUND: The COVID-19 pandemic caused by SARS-CoV-2 remains public health burdens and many unresolved issues worldwide. Molecular assays based on real-time RT-PCR are critical for the detection of SARS-CoV-2 in clinical specimens from patients suspected of COVID-19. OBJECTIVE: We aimed to establish and validate an in-house real-time RT-PCR for the detection of SARS-CoV-2. METHODOLOGY: Primers and probes sets in our in-house real-time RT-PCR assay were designed in conserved regions of the N and E target genes. Optimized multiplex real-time RT-PCR assay was validated using the first WHO International Standard (NIBSC code: 20/146) and evaluated clinical performance. RESULTS: The limit of detection validated using the first WHO International Standard was 159 IU/ml for both E and N target genes. The evaluation of clinical performance on 170 clinical samples showed a positive percent agreement of 100% and the negative percent agreement of 99.08% for both target genes. The Kappa value of 0.99 was an excellent agreement, the strong correlation of Ct values observed between two tests with r2  = 0.84 for the E gene and 0.87 for the N gene. Notably, we assessed on 60 paired saliva and nasopharyngeal samples. The overall agreement was 91.66%, and Kappa value of 0.74 showed a high agreement between two types of samples. When using nasopharyngeal swabs as the reference standard, positive percent agreement, and negative percent agreement were 91.83% and 90.90%, respectively. CONCLUSION: In the present study, we established and validated an in-house real-time RT-PCR for molecular detection of SARS-CoV-2 in a resource-limited country.

Platelet activation in rabbits with decompression sickness.

Platelets are the most easily altered type of peripheral blood cell in decompression sickness (DCS), which can feature decreased platelet count and the appearance of platelet microparticles in plasma. We hypothesized that DCS results in platelet activation in the bloodstream. The present study was carried out on 45 rabbits. The platelet count and concentration of plasma platelet markers were determined in 35 rabbits; the platelet shape was observed under scanning electron microscope in 10 rabbits. All indexes were collected at two points: 24 hours before the simulated dive and 30 minutes after the simulated dive. Platelet count decreased noticeably after DCS, from 380.10 ± 73.61 (G/L) to 330.23 ± 115.72 (G/L), a change of approximately -13.49 ± 25.57 (%). Platelet count was further decreased in the severe DCS group (a change of -45.99 ± 18.57%). Platelet count after DCS was proportional to the survival time of the rabbits after DCS. The concentration of two plasma platelet markers (PF4 and BTG) did not demonstrate statistically significant change at 30 minutes after DCS. However, platelet shape was changed, and the following features were observed: oblong, distortion, flattening shape, sticking together, mixing of membrane, and abundance of pseudopods with a 100- to 200-nm diameter. We conclude there is platelet activation in the bloodstream in cases of DCS.