Single-Cell Immunogenomic Approach Identified SARS-CoV-2 Protective Immune Signatures in Asymptomatic Direct Contacts of COVID-19 Cases.

The response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is largely impacted by the level of virus exposure and status of the host immunity. The nature of protection shown by direct asymptomatic contacts of coronavirus disease 2019 (COVID-19)-positive patients is quite intriguing. In this study, we have characterized the antibody titer, SARS-CoV-2 surrogate virus neutralization, cytokine levels, single-cell T-cell receptor (TCR), and B-cell receptor (BCR) profiling in asymptomatic direct contacts, infected cases, and controls. We observed significant increase in antibodies with neutralizing amplitude in asymptomatic contacts along with cytokines such as Eotaxin, granulocyte-colony stimulating factor (G-CSF), interleukin 7 (IL-7), migration inhibitory factor (MIF), and macrophage inflammatory protein-1α (MIP-1α). Upon single-cell RNA (scRNA) sequencing, we explored the dynamics of the adaptive immune response in few representative asymptomatic close contacts and COVID-19-infected patients. We reported direct asymptomatic contacts to have decreased CD4+ naive T cells with concomitant increase in CD4+ memory and CD8+ Temra cells along with expanded clonotypes compared to infected patients. Noticeable proportions of class switched memory B cells were also observed in them. Overall, these findings gave an insight into the nature of protection in asymptomatic contacts.

Detection of SARs-CoV-2 in wastewater using the existing environmental surveillance network: A potential supplementary system for monitoring COVID-19 transmission.

The ongoing COVID-19 pandemic is caused by SARs-CoV-2. The virus is transmitted from person to person through droplet infections i.e. when infected person is in close contact with another person. In January 2020, first report of detection of SARS-CoV-2 in faeces, has made it clear that human wastewater might contain this virus. This may illustrate the probability of environmentally facilitated transmission, mainly the sewage, however, environmental conditions that could facilitate faecal oral transmission is not yet clear. We used existing Pakistan polio environment surveillance network to investigate presence of SARs-CoV-2 using three commercially available kits and E-Gene detection published assay for surety and confirmatory of positivity. A Two-phase separation method is used for sample clarification and concentration. An additional high-speed centrifugation (14000Xg for 30 min) step was introduced, prior RNA extraction, to increase viral RNA yield resulting a decrease in Cq value. A total of 78 wastewater samples collected from 38 districts across Pakistan, 74 wastewater samples from existing polio environment surveillance sites, 3 from drains of COVID-19 infected areas and 1 from COVID 19 quarantine center drainage, were tested for presence of SARs-CoV-2. 21 wastewater samples (27%) from 13 districts turned to be positive on RT-qPCR. SARs-COV-2 RNA positive samples from areas with COVID 19 patients and quarantine center strengthen the findings and use of wastewater surveillance in future. Furthermore, sequence data of partial ORF 1a generated from COVID 19 patient quarantine center drainage sample also reinforce our findings that SARs-CoV-2 can be detected in wastewater. This study finding indicates that SARs-CoV-2 detection through wastewater surveillance has an epidemiologic potential that can be used as supplementary system to monitor viral tracking and circulation in cities with lower COVID-19 testing capacity or heavily populated areas where door-to-door tracing may not be possible. However, attention is needed on virus concentration and detection assay to increase the sensitivity. Development of highly sensitive assay will be an indicator for virus monitoring and to provide early warning signs.

Proliferation of SARS-CoV-2 B.1.1.7 Variant in Pakistan-A Short Surveillance Account.

The emergence of a more transmissible variant of SARS-CoV-2 (B1. 1.7) in the United Kingdom (UK) during late 2020 has raised major public health concerns. Several mutations have been reported in the genome of the B.1.1.7 variant including the N501Y and 69-70deletion in the Spike region that has implications on virus transmissibility and diagnostics. Although the B.1.1.7 variant has been reported by several countries, only three cases have been reported in Pakistan through whole-genome sequencing. Therefore, the objective of the study was to investigate the circulation of B.1.1.7 variant of concern (VOC) in Pakistani population. We used a two-step strategy for the detection of B.1.1.7 with initial screening through TaqPathTM COVID-19 CE-IVD RT-PCR kit (ThermoFisher Scientific, Waltham, US) followed by partial spike (S) gene sequencing of a subset of samples having the spike gene target failure (SGTF). From January 01, 2021, to February 21, 2021, a total of 2,650 samples were tested for SARS-CoV-2 and 70.4% (n = 1,867) showed amplification of all the 3 genes (ORF, N, and S). Notably, 29.6% (n=783) samples have been SGTF that represented numbers from all the four provinces and suggest a rather low frequency during the first 3 weeks of January (n = 10, n = 13, and n = 1, respectively). However, the numbers have started to increase in the last week of January, 2021. During February, 726 (93%) cases of SGTF were reported with a peak (n = 345) found during the 3rd week. Based on the partial sequencing of SGTF samples 93.5% (n = 29/31) showed the characteristic N501Y, A570D, P681H, and T716I mutations found in the B.1.1.7 variant. In conclusion, our findings showed an upsurge of B.1.1.7 cases in Pakistan during February, 2021 affecting 15 districts and warranting large scale genomic surveillance, strengthening of laboratory network and implementation of appropriate control measures in the country.

Foot and ankle trauma management during the COVID-19 pandemic: Experiences from a major trauma unit.

The COVID-19 pandemic has resulted in a paradigm shift in clinical practice, particularly in ways in which healthcare is accessed by patients and delivered by healthcare practitioners. Many of these changes have been serially modified in adaptation to growing service demands and department provision capacity. We evaluated the impact of the pandemic on the foot and ankle service at our trauma unit, assessing whether these adaptations to practice were justifiable, successful and sustainable for the future. This was a single-centre, retrospective cohort study analysing the patient care pathway from admission to discharge, for two pre-defined timeframes: Phase 0 (pre-lockdown phase) and Phase 1 (lockdown phase). Patients were split into stable and unstable injuries depending on their fracture pattern. The follow-up modality and duration were evaluated. Trauma throughput for the equivalent timeframe in 2019 was also analysed for comparison. There were 106 unstable fractures and 100 stable fractures in 2020.78 interventional procedures were performed on 72 patients with unstable fractures in Phase-1. Close contact casting was performed on 13 patients at presentation in the ED. Selective patients underwent partial fixation in theatre, which still provided adequate stability. 35% of patients with a stable fracture were discharged directly from the ED with written advice from a review letter. The treatment modality in selective patients, particularly the vulnerable should be carefully assessed. Interventions performed at presentation often negate the need for admission. Partial fixation reduces intraoperative time and surgical insult. Integrating telemedicine into the care pathway, particularly for stable ankle fractures reduces the need for physician-patient contact and eases follow-up burden. Many of our recommended changes are easily replicated in other clinical settings. Should these adaptations demonstrate long-term sustainability, it is likely they will remain incorporated into future clinical practice.

COVID-19 pandemic and breast cancer management: A retrospective observational clinical study from Pakistan.

BACKGROUND: COVID-19 outbreak has adversely affected care of breast cancer patients world-wide. There is paucity of available data on cancer management in lower-middle income countries during this pandemic, we sought to determine the institutional approach towards management of breast cancer patients and the outcomes during COVID-19 pandemic at our institution. MATERIALS AND METHODS: Clinicopathological and treatment record of cancer patients who presented to the Breast Clinic from 15th March to 31st December 2020 was retrieved from the institutional database for this retrospective clinical observational study. RESULTS: A total of 292 patients were qualified for the analysis in which 206 patients (70.5%) underwent breast cancer surgeries. Only 10 of them (4.9%) were identified to be COVID-19 virus positive on routine pre-operative RT-PCR test before elective surgeries. All were asymptomatic, received home-based care via telecommunication and were operated at a later date when test turned out negative. None of them developed any complications post-operatively. Another group of 86 patients (29.5%) were referred for Neoadjuvant/Systemic chemotherapy in which COVID-19 infection was detected in only 4 patients (4.6%) while receiving cycles. Two patients shown symptoms of cough and fever hence hospitalised but were not candidates for Intensive Care Unit admission while other two patient were asymptomatic and isolated at home. All patients recovered well and chemotherapy was commenced again after negative RT-PCR test. No mortality was observed. CONCLUSION: Despite of being a global crisis particularly for cancer patients we observed infectivity, complications and fatality much lower among breast cancer cases. Further research is needed in this regard including public and private sector hospitals for better understanding behavior of COVID-19 disease and achieve common goal to combat COVID-19 and cancer together.

Reverse transcriptase loop-mediated isothermal amplification (RT-LAMP)-based diagnosis: A potential alternative to quantitative real-time PCR based detection of the novel SARS-COV-2 virus.

The sudden outbreak of the novel Coronavirus infectious disease (COVID-19) resulted in significant challenges to global health systems. One of the primary challenges is rapid, reliable, and accurate detection of the severe acute respiratory syndrome coronavirus 2 (SARS-COV-2) virus among the suspected COVID-19-infected individuals. At present, quantitative real-time PCR (qRT-PCR) is a widely used diagnostic method. However, it requires expensive instruments and expertise in the interpretation of results. These constraints reflect the significant need for the development of alternative diagnostic options. This study will validate the use and efficiency of the reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) assay as a potential alternative for the detection of COVID-19. For this purpose, a cohort of 297 suspected COVID-19 patients was tested using both the RT-LAMP assay and the conventional RT-PCR method. For the RT-LAMP assay, three genes (orf-1ab, N, and S) were identified as the target sites for the detection of COVID-19. Based on a comparative assessment, 117 out of 124 positive COVID-19 cases were observed using the RT-LAMP technique with an overall 91.45% sensitivity. Interestingly, where a consensus on 163 individuals free of SARS-Cov-2 was observed, RT-LAMP specificity was 90%. Based on these findings, the robustness of the technique, and the reduced dependency on expensive instrumentation, RT-LAMP-based COVID-19 detection is strongly recommended as a potential alternative assay.