Validating the inactivation of viral pathogens with a focus on SARS-CoV-2 to safely transfer samples from high-containment laboratories.

Introduction: Pathogen leak from a high-containment laboratory seriously threatens human safety, animal welfare, and environmental security. Transportation of pathogens from a higher (BSL4 or BSL3) to a lower (BSL2) containment laboratory for downstream experimentation requires complete pathogen inactivation. Validation of pathogen inactivation is necessary to ensure safety during transportation. This study established a validation strategy for virus inactivation. Methods: SARS-CoV-2 wild type, delta, and omicron variants underwent heat treatment at 95°C for 10 minutes using either a hot water bath or a thermocycler. To validate the inactivation process, heat-treated viruses, and untreated control samples were incubated with A549-hACE2 and Vero E6-TMPRSS2-T2A-ACE2 cells. The cells were monitored for up to 72 hours for any cytopathic effects, visually and under a microscope, and for virus genome replication via RT-qPCR. The quality of post-treated samples was assessed for suitability in downstream molecular testing applications. Results: Heat treatment at 95°C for 10 minutes effectively inactivated SARS-CoV-2 variants. The absence of cytopathic effects, coupled with the inability of virus genome replication, validated the efficacy of the inactivation process. Furthermore, the heat-treated samples proved to be qualified for COVID-19 antigen testing, RT-qPCR, and whole-genome sequencing. Discussion: By ensuring the safety of sample transportation for downstream experimentation, this validation approach enhances biosecurity measures. Considerations for potential limitations, comparisons with existing inactivation methods, and broader implications of the findings are discussed.

Montelukast and Telmisartan as Inhibitors of SARS-CoV-2 Omicron Variant.

Earlier studies with montelukast (M) and telmisartan (T) have revealed their potential antiviral properties against SARS-CoV-2 wild-type (WT) but have not assessed their efficacy against emerging Variants of Concern (VOCs) such as Omicron. Our research fills this gap by investigating these drugs' impact on VOCs, a topic that current scientific literature has largely overlooked. We employed computational methodologies, including molecular mechanics and machine learning tools, to identify drugs that could potentially disrupt the SARS-CoV-2 spike RBD-ACE2 protein interaction. This led to the identification of two FDA-approved small molecule drugs, M and T, conventionally used for treating asthma and hypertension, respectively. Our study presents an additional potential use for these drugs as antivirals. Our results show that both M and T can inhibit not only the WT SARS-CoV-2 but also, in the case of M, the Omicron variant, without reaching cytotoxic concentrations. This novel finding fills an existing gap in the literature and introduces the possibility of repurposing these drugs for SARS-CoV-2 VOCs, an essential step in responding to the evolving global pandemic.

Receptor-Binding-Motif-Targeted Sanger Sequencing: a Quick and Cost-Effective Strategy for Molecular Surveillance of SARS-CoV-2 Variants.

Whole-genome sequencing (WGS) is the gold standard for characterizing the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genome and identification of new variants. However, the cost involved and time needed for WGS prevent routine, rapid clinical use. This study aimed to develop a quick and cost-effective surveillance strategy for SARS-CoV-2 variants in saliva and nasal swab samples by spike protein receptor-binding-motif (RBM)-targeted Sanger sequencing. Saliva and nasal swabs prescreened for the presence of the nucleocapsid (N) gene of SARS-CoV-2 were subjected to RBM-specific single-amplicon generation and Sanger sequencing. Sequences were aligned by CLC Sequence Viewer 8, and variants were identified based upon specific mutation signature. Based on this strategy, the present study identified Alpha, Beta/Gamma, Delta, and Omicron variants in a quick and cost-effective manner. IMPORTANCE The coronavirus disease 2019 (COVID-19) pandemic resulted in 427 million infections and 5.9 million deaths globally as of 21 February 2022. SARS-CoV-2, the causative agent of the COVID-19 pandemic, frequently mutates and has developed into variants of major public health concerns. Following the Alpha variant (B.1.1.7) infection wave, the Delta variant (B.1.617.2) became prevalent, and now the recently identified Omicron (B.1.1.529) variant is spreading rapidly and forming BA.1, BA.1.1, BA.2, BA.3, BA.4, and BA.5 lineages of concern. Prompt identification of mutational changes in SARS-CoV-2 variants is challenging but critical to managing the disease spread and vaccine/therapeutic modifications. Considering the cost involved and resource limitation of WGS globally, an RBM-targeted Sanger sequencing strategy is adopted in this study for quick molecular surveillance of SARS-CoV-2 variants.

Is abnormal sperm function an indicator among couples with recurrent pregnancy loss?

OBJECTIVE: To determine whether or not sperm function parameters are altered in male partners of couples with a history of idiopathic recurrent pregnancy loss (RPL). DESIGN: In comparison with proven fertile volunteers, sperm function parameters like hypo-osmotic swelling (HOS), acrosomal status (AS), and nuclear chromatin decondensation (NCD) were assessed in vitro from male partners of couples with a history of idiopathic RPL. SETTING: Infertility clinic and andrology laboratory at National Institute of Health and Family Welfare. PATIENT(S): Male partners of couples with a history of idiopathic RPL and proven fertile male volunteers (control). INTERVENTION(S): Standard semen analysis, assessment of sperm morphology, and sperm function with tests such as HOS, AS, and NCD. MAIN OUTCOME MEASURE(S): Sperm paameters, such as HOS, AS, and NCD, were assessed in semen samples from RPL in comparison with the proven fertile control group. RESULT(S): Semen samples from the idiopathic RPL group showed below normal test scores in 57.1% of the cases for all three sperm parameters. The highest aberration (83% of cases) in sperm attributes was observed in NCD, followed by AS (45.7%) and HOS (42.9%). In contrast, abnormality in sperm morphology was limited to 5.7% of the cases. Subnormal sperm function is directly proportional with subnormal sperm motility (<50%) in 23% of the cases. Even in semen samples with normal sperm motility, sperm function scores were below normal in 31.4% of the RPL group. CONCLUSION(S): Reduction in test scores of sperm function, like HOS, AS, and NCD, in male partners of couples with idiopathic RPL suggests that sperms with altered or lowered functional competencies, if they fertilize the oocytes, may lead to the development of an unsustainable embryo resulting in early pregnancy loss. Normal sperm motility does not always ensure normal sperm function scores.

Development of a rapid, sensitive, and reproducible laboratory test kit for the assessment of plasma membrane integrity of human sperm.

OBJECTIVE: To develop a rapid, sensitive, and reproducible hypoosmotic swelling test kit for the assessment of plasma membrane integrity of human sperm in vitro. DESIGN: Prospective comparison of results with the World Health Organization (WHO) method, performed simultaneously. SETTING: Infertility center in a major city in India. PATIENT(S): Couples who presented for infertility evaluation. INTERVENTION(S): The sperm tail-coiling pattern representing sperm plasma membrane integrity was analyzed by using different concentrations of NaCl and also with plain double distilled water (ddH(2)O). Hypoosmotic swelling solution with 2% NaCl in ddH(2)O equivalent to 68 Osm/L was selected for further analysis because it provided the highest and the qualitatively best type (g) of tail coiling among the various other options tried in comparison. MAIN OUTCOME MEASURE(S): A rapid laboratory test kit for the assessment of plasma membrane integrity of sperm was developed that is equally sensitive and reproducible as that described in the WHO protocol. The test was validated by using different normal and subnormal semen samples and in comparison with the standard WHO protocol. RESULT(S): After the screening with different hypoosmotic solutions by using semen samples from fertile volunteers, NaCl (2%) provided the highest and the best types of typical tail coiling, characteristic of sperm with good plasma membrane integrity. The study was then extended to 60 normozoospermic semen samples, which demonstrated a hypoosmotic swelling response of 69.5% +/- 5.23% and 71.5% +/- 4.89%, as per WHO or the present modified method, respectively (coefficient of correlation, r = 0.741). In 13 oligospermic and 18 teratozoospermic subjects, the hypoosmotic swelling response of sperm as per the new method were 36.4% +/- 5.75% and 34.2% +/- 7.78%, respectively, which were comparable to those obtained through the WHO method. There was a 15% drop in viability of sperm after the hypoosmotic challenge (5 min), irrespective of the hypoosmotic solution used. No other morphological alterations in sperm were observed after the hypoosmotic challenge. CONCLUSION(S): The new test kit can be used routinely in laboratories for assessment of plasma membrane integrity of sperm in vitro.