ABSTRACT
ObjectivesThe objective of the current study was to develop a lower-cost and scalable protocol to identify and monitor SARS-CoV-2 variants in Paraguay by pairing real-time RT-PCR detection of spike mutations with amplicon Sanger sequencing and whole-genome Nanopore sequencing. Methods201 acute-phase nasopharyngeal samples from SARS-CoV-2-positive individuals were tested with two rRT-PCRs: 1) N2RP assay to confirm SARS-CoV-2 RNA detection (CDC N2 target), and 2) the Spike SNP assay to detect mutations in the spike receptor binding domain. The assay was performed with probes to identify mutations associated with the following variants: alpha (501Y), beta/gamma (417variant/484K/501Y), delta (452R/478K), and lambda (452Q/490S). ResultsAll samples were positive for SARS-CoV-2 in the N2RP assay (mean Ct, 20.8; SD 5.6); 198/201 (98.5%) tested positive in the Spike SNP assay. The most common genotype was 417variant/484K/501Y, detected in 102/198 samples (51.5%) and most consistent with P.1 lineage (gamma variant) in Paraguay. No mutations (K417 only) were found in 64/198 (32.3%); and K417/484K was identified in 22/198 (11.1%), consistent with P.2 (zeta). Seven samples (3.5%) tested positive for 452R without 478K, and one sample with genotype K417/501Y was confirmed as B.1.1.7 (alpha). Results were confirmed by Sanger sequencing in 181/181 samples (100%) with high-quality amplicon sequences, and variant calls were consistent with Nanopore sequencing in 29/29 samples. ConclusionsThe Spike SNP assay provides accurate detection of mutations associated with SARS-CoV-2 variants. This can be implemented in laboratories performing rRT-PCR to improve population-level surveillance for these mutations and inform the judicious use of scarce sequencing resources.
ABSTRACT
Semen cryopreservation is a useful tool in assisted reproduction, which may have impact on sperm characteristics during freezing and thawing. The aim of this study was to assess the integrity of the acrosome and motility of cryopreserved and thawed spermatozoos in hyperviscous and no viscous samples. In semen samples spermiogram, glandular markers, acrosome integrity, culture and the levels markers accessory glands were measured. Each aliquot of semen was immersed in cryoprotectant and maintained in a commercial freezer at -196 ° C. After 30 days, these were thawed and in the cell pellet resuspended, spermatic motility and acrosomal integrity were evaluated. In thawed samples, there were significant decreases in progressive motility (p <0.05), vitality (p <0.005) and acrosome integrity (p <0.05) with respect to fresh sperm, this decline was most evident in hyperviscous samples. The viscosity of fresh semen was inversely related to motility and acrosome integrity before and after freezing (p <0.05). Twenty semen samples showed the presence of microorganisms and C. trachomatis IgA antibodies, of which fifteen showed hyperviscosity. Biochemical analysis demonstrated that semen samples with low levels of citric acid had less acrosomal integrity both before and after freezing (p <0.05). The viscoelasticity and citric acid levels are associated with prostate dysfunction, low sperm motility and premature acrosome reaction, which can reduce the fertilizing capacity of sperm. The etiology of hyperviscosity remains complex; however, to preserve motility and acrosome integrity, its causes must be investigated previously in the seminal samples to be subjected to cryopreservation.
