Human Nasopharyngeal Research Articles

Development and implementation of a core genome multilocus sequence typing scheme for Haemophilus influenzae.

Haemophilus influenzae is part of the human nasopharyngeal microbiota and a pathogen causing invasive disease. The extensive genetic diversity observed in H. influenzae necessitates discriminatory analytical approaches to evaluate its population structure. This study developed a core genome multilocus sequence typing (cgMLST) scheme for H. influenzae using pangenome analysis tools and validated the cgMLST scheme using datasets consisting of complete reference genomes (N = 14) and high-quality draft H. influenzae genomes (N = 2297). The draft genome dataset was divided into a development dataset (N = 921) and a validation dataset (N = 1376). The development dataset was used to identify potential core genes, and the validation dataset was used to refine the final core gene list to ensure the reliability of the proposed cgMLST scheme. Functional classifications were made for all the resulting core genes. Phylogenetic analyses were performed using both allelic profiles and nucleotide sequence alignments of the core genome to test congruence, as assessed by Spearman's correlation and ordinary least square linear regression tests. Preliminary analyses using the development dataset identified 1067 core genes, which were refined to 1037 with the validation dataset. More than 70% of core genes were predicted to encode proteins essential for metabolism or genetic information processing. Phylogenetic and statistical analyses indicated that the core genome allelic profile accurately represented phylogenetic relatedness among the isolates (R 2 = 0.945). We used this cgMLST scheme to define a high-resolution population structure for H. influenzae, which enhances the genomic analysis of this clinically relevant human pathogen.

Differential expression of host oncogenes in human papillomavirus-associated nasopharyngeal and cervical epithelial cancers.

Human papillomavirus (HPV)-related cervical and nasopharyngeal cancers differ in molecular mechanisms underlying the oncogenic processes. The disparity may be attributed to differential expression of oncoproteins. The current study investigated the host oncogenes expression pattern in HPV-associated cervical and nasopharyngeal cancer. Formalin-fixed paraffin-embedded tissues originating from the nasopharyngeal and cervical regions were screened using Hematoxylin and Eosin staining. Genomic DNA and total RNA were extracted from confirmed cancer biopsies and non-cancer tissues (NC). HPV was detected by PCR using MY09/GP5+/6+ primers. Protein expression levels of AKT, IQGAP1, and MMP16 in HPV-infected cancers and controls were determined by immunohistochemistry. RT-qPCR was used to profile mRNAs of the oncogenes. AKT and IQGAP1 proteins were highly expressed in the epithelial cancers compared with the non-cancer tissues (p < 0.05). IQGAP1 and MMP16 mRNAs level was significantly higher in the cancers than in the NC (p < 0.05), but not AKT mRNA levels. MMP16 protein was ubiquitously expressed in all tissues. AKT mRNA level was significantly elevated in CC compared with NPC (p < 0.001). However, the difference in AKT, IQGAP1 and MMP16 proteins level between CC and NPC was not significant (p > 0.05). The oncoproteins expression level between the HPV-positive and HPV-negative cancer biopsies showed no significant difference (p < 0.05). Current study reports AKT but not IQGAP1 and MMP16 mRNAs differentially expression in cervical and nasopharyngeal cancers, independent of HPV infection status.

Advancing SERS Diagnostics in COVID‐19 with Rapid, Accurate, and Label‐Free Viral Load Monitoring in Clinical Specimens via SFNet Enhancement

AbstractThis study presents an integrated approach combining surface‐enhanced Raman spectroscopy (SERS) with a specialized deep learning algorithm, SFNet, to offer a rapid, accurate, and label‐free alternative for COVID‐19 diagnosis and viral load quantification. The SiO2‐coated silver nanorod arrays are employed as the SERS substrates, fabricated using a reliable and effective glancing angle deposition technique. A dataset of 4800 SERS spectra from 120 positive and 120 negative inactivated clinical human nasopharyngeal swabs are collected directly on the SERS substrates without any labels. A SFNet algorithm is tailored to adapt to the unique spectral features inherent to SERS data, achieving a test accuracy of 98.5% and a blind test accuracy of 99.04%. Moreover, an optimized SFNet algorithm unveils the capability of estimating SARS‐CoV‐2 viral loads, accurately predicting the cycle threshold values (Ct values) of the three vital gene fragments with a root mean square error (RMSE) of 1.627 (1.3 for blind test). The methodology is substantiated using actual clinical specimens and completed in &lt;15 min, thereby strengthening its real‐world point‐of‐care applicability. This rapid and precise yet label‐free modality competes favorably with classical reverse‐transcription real‐time polymerase chain reaction (RT‐PCR) and marks an advancement in SERS‐based sensor algorithms.

Accurate detection of influenza A virus by use of a novel cross-priming isothermal amplification-based point-of-care assay.

Influenza virus is known to cause respiratory tract infections of varying severity in individuals of all ages. The EasyNAT Rapid Flu assay is a newly developed in vitro diagnostic test that employs cross-priming isothermal amplification (CPA) to detect and differentiate influenza A and B viruses in human nasopharyngeal (NP) swabs. The aim of this study is to determine the performance characteristics of the EasyNAT Rapid Flu assay for rapid detection of influenza virus. The limit of detection (LOD) and cross-reactivity of the EasyNAT Rapid Flu assay were assessed. The clinical performance of the assay was evaluated using NP swab samples that were tested with real-time reverse-transcription polymerase chain reaction (RT-PCR) and Xpert Xpress Flu/RSV assay. The LOD for the detection of influenza A and B using the EasyNAT Rapid Flu assay was found to be 500 copies/mL. Furthermore, the assay exhibited no cross-reactivity with other common respiratory viruses tested. For the 114 NP swab samples tested for influenza A using both the EasyNAT Rapid Flu assay and real-time RT-PCR, the two assays demonstrated a high level of agreement (κ = 0.963, P < 0.001), with a positive percentage agreement (PPA) of 97.7% and a negative percentage agreement (NPA) of 98.6%. Similarly, for the 43 NP swab samples tested for influenza A and B using both the EasyNAT Rapid Flu assay and Xpert Xpress Flu/RSV assay, the two assays showed a high level of agreement (κ = 0.933, P < 0.001), with the overall rate of agreement (ORA) of 97.7% for influenza A and 100% for influenza B. The EasyNAT Rapid Flu assay demonstrates excellent performance in the detection of influenza A, highlighted by its strong agreement with RT-PCR-based assays.IMPORTANCEThe newly developed EasyNAT Rapid Flu assay is an innovative cross-priming isothermal amplification-based method designed for detecting influenza A and B viruses at point-of-care settings. This study aims to thoroughly assess the analytical and clinical performance of the assay, offering valuable insights into its potential advantages and limitations. The findings of this research hold significant implications for clinical practice.

A cross-sectional study on the nasopharyngeal microbiota of individuals with SARS-CoV-2 infection across three COVID-19 waves in India.

Multiple variants of the SARS-CoV-2 virus have plagued the world through successive waves of infection over the past three years. Independent research groups across geographies have shown that the microbiome composition in COVID-19 positive patients (CP) differs from that of COVID-19 negative individuals (CN). However, these observations were based on limited-sized sample-sets collected primarily from the early days of the pandemic. Here, we study the nasopharyngeal microbiota in COVID-19 patients, wherein the samples have been collected across the three COVID-19 waves witnessed in India, which were driven by different variants of concern. The nasopharyngeal swabs were collected from 589 subjects providing samples for diagnostics purposes at the Centre for Cellular and Molecular Biology (CSIR-CCMB), Hyderabad, India and subjected to 16s rRNA gene amplicon - based sequencing. We found variations in the microbiota of symptomatic vs. asymptomatic COVID-19 patients. CP showed a marked shift in the microbial diversity and composition compared to CN, in a wave-dependent manner. Rickettsiaceae was the only family that was noted to be consistently depleted in CP samples across the waves. The genera Staphylococcus, Anhydrobacter, Thermus, and Aerococcus were observed to be highly abundant in the symptomatic CP patients when compared to the asymptomatic group. In general, we observed a decrease in the burden of opportunistic pathogens in the host microbiota during the later waves of infection. To our knowledge, this is the first analytical cross-sectional study of this scale, which was designed to understand the relation between the evolving nature of the virus and the changes in the human nasopharyngeal microbiota. Although no clear signatures were observed, this study shall pave the way for a better understanding of the disease pathophysiology and help gather preliminary evidence on whether interventions to the host microbiota can help in better protection or faster recovery.

Rapid and label-free screening of SARS-CoV-2 virus in the infected human nasopharyngeal swab samples using UiO-66-modified SPR chip

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) virus has become a dangerous human pathogen and caused severe illness that can lead to death. Yet, the standard practical diagnostic tools for detecting the SARS-CoV-2 virus require multi-step assay, including extraction and labeling steps. Thus, a simple and accurate detection tool is incredibly desirable to support on-site screening, preventing its spread. In this work, we developed a portable surface plasmon resonance (SPR) sensor chip modified with metal–organic frameworks (MOF) UiO-66 that serves as an immobilization matrix of single-chain variable fragment (scFv) receptors. Large surface area and good adsorption of UiO-66/scFv-modified surface lead to an improvement of binding capacity towards the protein target of the SARS-CoV-2 receptor binding domain (RBD). We further successfully demonstrated the use of this portable SPR to detect the SARS-CoV-2 virus from patients' nasopharyngeal swab samples. Interestingly, the SPR sensors can directly distinguish the positive and negative patients within 15 minutes on 23 samples, simultaneously, without the need for labeling, and the results are in line with the gold standard PCR tests. In addition, the detection limit of the sensors is 10.085 × 105 virus/mL, which follows the standard of practical screening tools. Therefore, these UiO-66 modified SPR chip sensors offer a rapid, label-free, and low-cost detection of the SARS-CoV-2 virus that can be potentially developed for other infectious diseases.

Mechanism of lncRNA FOXD3-AS1/miR-338-3p in the malignant progression of nasopharyngeal carcinoma through ceRNA.

This work aimed to analyze the impact of lncRNA FOXD3-AS1 (F-A) on the regulation of miR-338-3p (M) on the proliferation (Pro), migration (Mig), and invasion (Inv) of nasopharyngeal carcinoma (NPC) cells. F-A and M levels in human normal nasopharyngeal (NNP) NP69 cells and NPC CNE1 and CNE2 cells were detected by real-time quantitative PCR. After transfection or cotransfection with F-A shRNA, miR-338-3p mimic (MM), and miR-338-3p inhibitor (MI), cell Pro, Mig, and Inv (PMI) were detected using CCK-8, scratch healing, and Transwell chamber experiments, respectively. As a result, relative to NP69 cells, F-A was upregulated in CNE1 and CNE2 cells, while M was downregulated (P<0.01). F-A in CNE1 and CNE2 cells was downregulated and M was upregulated relative to shF-A (P<0.01). Furthermore, shF-A and MM groups demonstrated drastically reduced cell proliferative activity, Mig, and Inv versus CNE1/CNE2 groups. The cell proliferative activity, Mig, and Inv number of the MI group increased substantially (P<0.01). The shF-A+MM group exhibited markedly reduced cell proliferative activity, Mig, and cell number of Inv relative to those of CNE1/CNE2 (P<0.01). Moreover, the shF-A+MI group exhibited greatly increased cell proliferative activity, Mig, and cell number of Inv versus the shF-A+MM group (P<0.01). In short, lncRNA F-A level was abnormally upregulated, and that of M was downregulated in NPC. Interfering with lncRNA F-A level can upregulate M expression. Silencing of lncRNA F-A can inhibit PMI of NPC cells.

Analytical validation of quantitative SARS-CoV-2 subgenomic and viral load laboratory developed tests conducted on the Panther Fusion® (Hologic) with preliminary application to clinical samples.

Validate the performance characteristics of two analyte specific, laboratory developed tests (LDTs) for the quantification of SARS-CoV-2 subgenomic RNA (sgRNA) and viral load on the Hologic Panther Fusion® using the Open Access functionality. Custom-designed primers/probe sets targeting the SARS-CoV-2 Envelope gene (E) and subgenomic E were optimized. A 20-day performance validation following laboratory developed test requirements was conducted to assess assay precision, accuracy, analytical sensitivity/specificity, lower limit of detection and reportable range. Quantitative SARS-CoV-2 sgRNA (LDT-Quant sgRNA) assay, which measures intermediates of replication, and viral load (LDT-Quant VLCoV) assay demonstrated acceptable performance. Both assays were linear with an R2 and slope equal to 0.99 and 1.00, respectively. Assay precision was evaluated between 4-6 Log10 with a maximum CV of 2.6% and 2.5% for LDT-Quant sgRNA and LDT-Quant VLCoV respectively. Using negative or positive SARS-CoV-2 human nasopharyngeal swab samples, both assays were accurate (kappa coefficient of 1.00 and 0.92). Common respiratory flora and other viral pathogens were not detected and did not interfere with the detection or quantification by either assay. Based on 95% detection, the assay LLODs were 729 and 1206 Copies/mL for the sgRNA and VL load LDTs, respectively. The LDT-Quant sgRNA and LDT-Quant VLCoV demonstrated good analytical performance. These assays could be further investigated as alternative monitoring assays for viral replication; and thus, medical management in clinical settings which could inform isolation/quarantine requirements.

Pneumococcal bacteraemia in adults over a 10-year period (2011-2020): a clinical and serotype analysis.

Streptococcus pneumoniae (pneumococcus) is a human nasopharyngeal tract coloniser responsible for invasive pneumococcal disease, which is largely vaccine preventable. Vaccination is recommended from birth for all, and through adulthood for those with risk conditions. To describe the clinical and serotype analysis of pneumococcus bacteraemia over a 10-year period. A 10-year (February 2011-December 2020) retrospective review was performed on all adult (age ≥18 years) pneumococcus bacteraemia presenting to the four public hospitals in Western Sydney, Australia. Comorbidities and risk factors were recorded. Three hundred unique episodes of S. pneumoniae bloodstream infection (SPBI) were identified during the study period. The median age for SPBI was 63 years with 31.7% aged 70 years or older. A 94.7% had one or more risks factors for SPBI. Pneumonia was reported in 80% of all SPBI, whereas meningitis was reported in 6% and infective endocarditis in <1%. Asplenia was noted in 2.4%. Seven- and 30-day mortality was 6.6% and 11.9%, with a higher 30-day mortality in those aged ≥70 years (24.4%). The serotype distribution showed 7-valent conjugate vaccine covered 11.0% of all isolates, whereas 13-valent conjugate vaccine (13vPCV) and a 23-valent polysaccharide vaccine (23vPPV) covered 41.7% and 69.0% respectively. Immunisation details were available for 110 individuals, of whom, only 7.3% had received pneumococcal vaccination. Most patients with pneumococcal bacteraemia had age- or comorbidity-related risk factors but were not vaccinated. Two-thirds of cases occurred in people aged <70 years. 13vPCV and 23vPPV covered 41.7% and 69.0% of bacteraemic isolates.

A cross-sectional study of avian influenza A virus in Myanmar live bird markets: Detection of a newly introduced H9N2?

Zoonotic influenza surveillance in Myanmar is sparse, despite the risks of introduction of such viruses from neighboring countries that could impact the poultry industry and lead to spillover to humans. In July and August 2019, our multi-institutional partnership conducted a One Health-oriented, cross-sectional surveillance (weekly for 3 weeks) for influenza A and influenza D viruses at the three largest live bird markets in Yangon, Myanmar. The 27 bioaerosols, 90 bird cage swabs, 90 bird oropharyngeals, and 90 human nasopharyngeal samples yielded molecular influenza A detections in 8 bioaerosols (30.0%), 16 bird cages (17.8%), 15 bird oropharyngeals (16.7%), and 1 human nasopharyngeal (1.1%) samples. No influenza D was detected. Seven of the influenza A virus detections were found to be subtype A/H9N2, and one human nasopharyngeal sample was found to be subtype A/H1pdm. Among all IAV-positive samples, three of the A/H9N2-positive samples yielded live viruses from egg culture and their whole genome sequences revealing they belonged to the G9/Y280 lineage of A/H9N2 viruses. Phylogenetic analyses showed that these A/H9N2 sequences clustered separately from A/H9N2 viruses that were previously detected in Myanmar, supporting the notion that A/H9N2 viruses similar to those seen in wider Southeast Asia may have been introduced to Myanmar on multiple occasions. These findings call for increased surveillance efforts in Myanmar to monitor for the introduction of novel influenza viruses in poultry, as well as possible reassortment and zoonotic virus transmission.

In-situ fabrication of 3D interior hotspots templated with a protein@Au core–shell structure for label-free and on-site SERS detection of viral diseases

Nanoscale plasmonic hotspots play a critical role in the enhancement of molecular Raman signals, enabling the sensitive and reliable trace analysis of biomedical molecules via surface-enhanced Raman spectroscopy (SERS). However, effective and label-free SERS diagnoses in practical fields remain challenging because of clinical samples' random adsorption and size mismatch with the nanoscale hotspots. Herein, we suggest a novel SERS strategy for interior hotspots templated with protein@Au core–shell nanostructures prepared via electrochemical one-pot Au deposition. The cytochrome c and lysates of SARS-CoV-2 (SLs) embedded in the interior hotspots were successfully functionalized to confine the electric fields and generate their optical fingerprint signals, respectively. Highly linear quantitative sensitivity was observed with the limit-of-detection value of 10−1 PFU/mL. The feasibility of detecting the targets in a bodily fluidic environment was also confirmed using the proposed templates with SLs in human saliva and nasopharyngeal swabs. These interior hotspots templated with the target analytes are highly desirable for early and on-site SERS diagnoses of infectious diseases without any labeling processes.

Epstein-Barr viral product-containing exosomes promote fibrosis and nasopharyngeal carcinoma progression through activation of YAP1/FAPα signaling in fibroblasts

BackgroundThe progression of nasopharyngeal carcinoma (NPC) is profoundly affected by Epstein-Barr virus (EBV) infection. However, the role of EBV in the intercommunication between NPC and surrounding stromal cells has yet to be explored.MethodsNPC biopsies were obtained for immunohistochemical (IHC) analyses. Clinical correlations between the expression of active YAP1/FAPα and the fibrotic response and between YAP1/FAPα and the density of cytotoxic CD8a+ T lymphocytes were determined. Survival times based on IHC scores were compared between groups using Kaplan-Meier survival and log-rank tests. Independent prognostic factors for metastasis/recurrence-free survival and overall survival were identified using univariate and multivariate Cox regression models. Fibroblasts were isolated from human nasopharyngeal biopsies. Exosomes were purified from culture supernatants of EBV+-positive NPC cells. The effects of EBV product-containing exosomes on fibroblast activation, fibrotic response, tumor growth, immune response, and correlations between the expression of featured genes were investigated using gel contraction assays, ELISAs, EdU incorporation assays, real-time impedance assays, RNA sequencing, immunostaining, 3D cancer spheroid coculture systems, and an NPC xenograft model.ResultsNPC patients who developed metastasis had significantly higher levels of active YAP1 and FAPα in their tumor stroma, which was further correlated with tumor fibrosis and poorer metastasis-free survival. Exosomes released from EBV+-NPC cells contained abundant FAPα protein and EBV-encoded latent membrane protein 1. Viral product-containing exosomes markedly enhanced the fibrotic response and tumor growth in a mouse xenograft model. IHC analyses of human NPC and NPC xenografts revealed positive correlations between levels of active YAP1 and FAPα, YAP1 and the fibrotic response, and FAPα and the fibrotic response. Mechanistic studies showed that treatment of fibroblasts with viral product-containing exosomes promoted the characteristics of cancer-associated fibroblasts by stimulating YAP1 signaling and the production of the immunosuppressive cytokines IL8, CCL2, and IL6. Inhibition of YAP1 activation markedly reversed these exosome-mediated protumoral effects, resulting in reduced contractility, inactivation of YAP1 signaling, and decreased production of immunosuppressive cytokines in fibroblasts. Furthermore, fibroblasts stimulated with these viral product-containing exosomes promoted NPC resistance to T cell-mediated cytotoxicity within tumor spheroids. In NPC tissues, a significant negative correlation was found between YAP1/FAPα and the density of CD8a+ T lymphocytes with a granzyme B signature.ConclusionEBV orchestrates interactions with the host and surrounding stroma by stimulating the functions of YAP1 and FAPα in fibroblasts through exosome cargos to create a more immunosuppressive, proinvasive microenvironment.

Analysis of SARS-CoV-2 known and novel subgenomic mRNAs in cell culture, animal model, and clinical samples using LeTRS, a bioinformatic tool to identify unique sequence identifiers.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has a complex strategy for the transcription of viral subgenomic mRNAs (sgmRNAs), which are targets for nucleic acid diagnostics. Each of these sgmRNAs has a unique 5′ sequence, the leader–transcriptional regulatory sequence gene junction (leader–TRS junction), that can be identified using sequencing. High-resolution sequencing has been used to investigate the biology of SARS-CoV-2 and the host response in cell culture and animal models and from clinical samples. LeTRS, a bioinformatics tool, was developed to identify leader–TRS junctions and can be used as a proxy to quantify sgmRNAs for understanding virus biology. LeTRS is readily adaptable for other coronaviruses such as Middle East respiratory syndrome coronavirus or a future newly discovered coronavirus. LeTRS was tested on published data sets and novel clinical samples from patients and longitudinal samples from animal models with coronavirus disease 2019. LeTRS identified known leader–TRS junctions and identified putative novel sgmRNAs that were common across different mammalian species. This may be indicative of an evolutionary mechanism where plasticity in transcription generates novel open reading frames, which can then subject to selection pressure. The data indicated multiphasic abundance of sgmRNAs in two different animal models. This recapitulates the relative sgmRNA abundance observed in cells at early points in infection but not at late points. This pattern is reflected in some human nasopharyngeal samples and therefore has implications for transmission models and nucleic acid–based diagnostics. LeTRS provides a quantitative measure of sgmRNA abundance from sequencing data. This can be used to assess the biology of SARS-CoV-2 (or other coronaviruses) in clinical and nonclinical samples, especially to evaluate different variants and medical countermeasures that may influence viral RNA synthesis.

SARS-CoV-2 detection using a nanobody-functionalized voltammetric device

BackgroundAn ongoing need during the COVID-19 pandemic has been the requirement for accurate and efficient point-of-care testing platforms to distinguish infected from non-infected people, and to differentiate SARS-CoV-2 infections from other viruses. Electrochemical platforms can detect the virus via its envelope spike protein by recording changes in voltammetric signals between samples. However, this remains challenging due to the limited sensitivity of these sensing platforms.MethodsHere, we report on a nanobody-functionalized electrochemical platform for the rapid detection of whole SARS-CoV-2 viral particles in complex media such as saliva and nasopharyngeal swab samples. The sensor relies on the functionalization of gold electrode surface with highly-oriented Llama nanobodies specific to the spike protein receptor binding domain (RBD). The device provides results in 10 min of exposure to 200 µL of unprocessed samples with high specificity to SARS-CoV-2 viral particles in human saliva and nasopharyngeal swab samples.ResultsThe developed sensor could discriminate between different human coronavirus strains and other respiratory viruses, with 90% positive and 90% negative percentage agreement on 80 clinical samples, as compared to RT-qPCR.ConclusionsWe believe this diagnostic concept, also validated for RBD mutants and successfully tested on Delta variant samples, to be a powerful tool to detect patients’ infection status, easily extendable to other viruses and capable of overcoming sensing-related mutation effects.

Fluorescent bioassay for SARS-CoV-2 detection using polypyrene-g-poly(ε-caprolactone) prepared by simultaneous photoinduced step-growth and ring-opening polymerizations.

The construction of a rapid and easy immunofluorescence bioassay for SARS-CoV-2 detection is described. We report for the first time a novel one-pot synthetic approach for simultaneous photoinduced step-growth polymerization of pyrene (Py) and ring-opening polymerization of ε-caprolactone (PCL) to produce a graft fluorescent copolymer PPy-g-PCL that was conjugated to SARS-CoV-2-specific antibodies using EDC/NHS chemistry. The synthesis steps and conjugation products were fully characterized using standard spectral analysis. Next, the PPy-g-PCL was used for the construction of a dot-blot assay which was calibrated for applications to human nasopharyngeal samples. The analytical features of the proposed sensor showed a detection range of 6.03–8.7 LOG viral copy mL−1 (Ct Scores: 8–25), the limit of detection (LOD), and quantification (LOQ) of 1.84 and 6.16 LOG viral copy mL−1, respectively. The repeatability and reproducibility of the platform had a coefficient of variation (CV) ranging between 1.2 and 5.9%. The fluorescence-based dot-blot assay was tested with human samples. Significant differences were observed between the fluorescence intensity of the negative and positive samples, with an overall correct response of 93.33%. The assay demonstrated a high correlation with RT-PCR data. This strategy opens new insights into simplified synthesis procedures of the reporter molecules and their high potential sensing and diagnosis applications.Graphical abstract Supplementary InformationThe online version contains supplementary material available at 10.1007/s00604-022-05244-2.

Pandemic Situation in Pakistan: Recoveries and Mortalities Rate of COVID-19 during First and Second Waves of Pandemic in Pakistan - Data from Feb to Dec 2020

The novel coronavirus disease COVID-19 outbreak has caused significant global issues and challenged healthcare systems across the globe and in Pakistan as well. This infectious disease is found to be a pandemic and has massively impacted countries across the world. The disease is still rapidly spreading and has caused many deaths in Pakistan, according to the world health organization; early in the year of 2020, Pakistan was ranked among the top 10 countries in the world reporting the highest number of new cases of COVID-19 per day during the first wave of the outbreak. The second wave of COVID-19 was relatively high on the first wave of disease in the current scenario of the pandemic. In the all over Pakistan, the real-time PCR is being applied for the qualitative detection of COVID-19 in the human nasopharyngeal swab specimen. From Feb to Dec.2020, in Pakistan there were a total of 416,499 cases registered across the country there have been (2%) deaths, with (85.2%) total recoveries. Sindh and Punjab were the most affected provinces; however, Balochistan, KPK, and other regions of Pakistan were less affected comparatively. This short communication is carried out to shed light on the coronavirus outbreak in the country; it will help to highlight the recoveries and mortalities rate of first and second waves of the disease.

Evaluation of long-term stability of SARS-CoV-2 nucleic acid extracted from human nasopharyngeal samples

The maintenance of SARS-Cov-2 RNA samples poses a new challenge for laboratories and researchers. In addition, it is a requirement in order to identify what strain of the new coronavirus is predominant in a region, for instance. Therefore, it is a must to keep the quality and viability of stored RNA to respond to this and other valid questions. In other to test the quality of our samples and storing protocols, we randomly checked RNA samples four different times over one year using a second RT-PCR assay after the first test. The virus genes, N1 and N2, showed no significant increase in the media of the CT value between the first assay and subsequent times with p > 0.05. However, the human RP gene showed differences in the first three times analyzed, but within the acceptable sample cut-off, according to the test manufacturer. After one year, the RNA extracted from human nasopharyngeal specimens are viable to detect the virus SARS-CoV-2 genes with minor changes.

Improving the specificity of nucleic acid detection with endonuclease-actuated degradation

Nucleic acid detection is essential for numerous biomedical applications, but often requires complex protocols and/or suffers false-positive readouts. Here, we describe SENTINEL, an approach that combines isothermal amplification with a sequence-specific degradation method to detect nucleic acids with high sensitivity and sequence-specificity. Target single-stranded RNA or double-stranded DNA molecules are amplified by loop-mediated isothermal amplification (LAMP) and subsequently degraded by the combined action of lambda exonuclease and a sequence-specific DNA endonuclease (e.g., Cas9). By combining the sensitivity of LAMP with the precision of DNA endonucleases, the protocol achieves attomolar limits of detection while differentiating between sequences that differ by only one or two base pairs. The protocol requires less than an hour to complete using a 65 °C heat block and fluorometer, and detects SARS-CoV-2 virus particles in human saliva and nasopharyngeal swabs with high sensitivity.

A new perspective on ancient Mitis group streptococcal genetics

Mitis group Streptococcus are human obligate bacteria residing in the nasopharynx and oral cavity. They comprise both commensal and pathogenic species with the most well-known being Streptococcus pneumoniae – a leading cause of meningitis and pneumonia. A primary difference between the commensal and pathogenic species is the presence of the polysaccharide capsule – a major virulence factor in S. pneumoniae , also present in other commensal species. Our current understanding of the evolutionary divergence of the pathogenic and commensal species has been inferred from extant strains. Ancient genomes can further elucidate streptococcal evolutionary history. We extracted streptococcal genome reads from a 5700-year-old ancient metagenome and worked towards characterizing them. Due to excessive within- and between-species recombination common among streptococci we were unable to parse individual species. Further, the composite reads of the ancient metagenome do not fit within the diversity of any specific extant species. Using a capsular gene database and AT-content analysis we determined that this ancient metagenome is missing polysaccharide synthesis genes integral to streptococcal capsule formation. The presence of multiple zinc metalloproteases suggests that adaptation to host IgA1 had begun and the presence of other virulence factors further implies development of close host–microbe interactions, though the absence of a capsule suggests an inability to cause invasive disease. The presence of specific virulence factors such as pneumolysin implies stable maintenance of such genes through streptococcal evolution that may strengthen their value as anti-pneumococcal vaccine antigens, while maintaining awareness of their potential presence in commensal species. Following from Jensen et al.’s initial analysis we provide historical context for this long time human nasopharyngeal resident, the Mitis group Streptococcus .

Licoflavanone exerts anticancer effects on human nasopharyngeal cancer cells via caspase activation, suppression of cell migration and invasion, and inhibition of m-TOR/PI3K/AKT pathway

Purpose: To study the anticancer effect of licoflavanone against human nasopharyngeal HKI carcinoma, and the mechanism involved.&#x0D; Methods: The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay was used to determine the effect of licoflavanone on cell viability, while DAPI staining and western blotting were used to study its proapoptotic effect. Morphological examination was performed under phase contrast microscopy. Transwell chamber assays were used to study cell migration and invasion. The expression levels of mTOR/PI3K/AKT signal pathway-related proteins were assayed by Western blotting.&#x0D; Results: Licoflavanone markedly suppressed the proliferation of nasopharyngeal HK1 cancer cells in a concentration-reliant pattern (p &lt; 0.01). The anticancer effects of licoflavanone were mediated via induction of pro-apoptotic effects and blocking of mTOR/PI3K/AKT signal pathway. Licoflavanone enhanced the activities of caspase-3, caspase-8, caspase-9 and cleaved caspase-3, as well as Bax and Bad. Moreover, licoflavanone blocked the migration and invasion of HK1 nasopharyngeal cancer cells.&#x0D; Conclusion: Licoflavanone exerts potent anticancer effects on human nasopharyngeal cancer cells via caspase activation, inhibition of cell migration and cell invasion, and down-regulation of m-TOR/PI3K/AKT signal pathway. Therefore, licoflavanone may be a useful lead drug for the development of a treatment strategy for nasopharyngeal cancer.