Comparison of longitudinal SARS-CoV-2 nasopharyngeal specimens reveals the transcriptomic COVIDome

Introduction/Objective SARS-Cov-2 is well established to introduce a cytokine-like storm among select individuals that results in multisystem failure and death. Comorbidities, age, oxygen status, and real-time appraisal of inflammatory markers in the blood have been used to risk stratify patients, however, these clinical markers do not comprehensively characterize the at-risk population or disease course. To understand the molecular underpinnings of the primary site of SARS-CoV-2 infection, here, we interrogated the transcriptomic profile of the nasopharyngeal tissue among paired SARS-CoV-2 specimens. Methods/Case Report We performed ribosomal depletion RNAseq on 24 primary samples, including 16 paired samples from 8 unique patients who converted between SARS-CoV-2 negative and positive status via clinical diagnostic qRT-PCR. Additional targeted qRT-PCR was performed for ACE2 and TMPRSS2 in an extension sample of 54 paired specimens from 27 unique patients who converted in their SARS-CoV-2 status on the basis of the qRT- PCR test. Differential gene expression, differential correlative expression with ACE2, and correlative expression with viral load was used to identify genes, which were integral to SARS-CoV-2 pathogenesis, so termed the COVIDome. Gene ontologies, pathways, and reactive infiltrate was assessed between specimens and compared with measures of clinical outcome using regression with appropriate correction for multiple hypotheses. Results (if a Case Study enter NA) We observed significant enrichment for ontologies of lymphocyte activation, specifically interferon gamma signaling; (P<1E-20) and platelet activation (P<1E-5). Genes specifically enriched across all three modules included: ADAMDEC1, EPSTI1, GRIP2, IRF7, KLHDC7B, OAS3, OASL, PIK3R4, RSAD2, and XAF1. Using CIBERSORT to approximate immune cell populations from bulk RNA, we observed and enrichment for CD4 immune cells, which was associated with viral status (P<0.01) while high-risk gene signatures were associated with measures of clinical outcome (P<0.05). Conclusion We characterized the pathogenesis of SARS-CoV-2 in longitudinal nasopharyngeal samples of COVID- 19 patients and related these molecular manifestations with measures of clinical outcome. As proof of principal, our findings suggest additional study in a large, longitudinal extension sample is warranted to validate and assess molecular features of clinical outcome associated with SARS-CoV-2 infection.

A first case report of nasopharyngeal Mycobacterium abscessus subspecies massiliense infection

Background Mycobacterium abscessus subspecies massiliense is a non-tuberculous mycobacteriosis and was subdivided from Mycobacterium abscessus in 2006. This article is the first report on nasopharyngitis caused by Mycobacterium abscessus subspecies massiliense. Case presentation A 45-year-old woman had an 18-month history of recurrent nasopharyngitis and presented with pain in the throat. Mycobacterial tissue culture and polymerase chain reaction testing revealed the presence of Mycobacterium abscessus subspecies massiliense in the nasopharyngeal tissue. This patient underwent surgery, followed by multiple rounds of chemotherapy with oral and intravenous antibiotic agents for 16 weeks. She has had no recurrence during the 56 weeks since treatment. Conclusion It is difficult to detect the presence of Mycobacterium abscessus subspecies massiliense in a culture from the swabbing sample. The tissue culture from a biopsy specimen is mandatory for the identification of the species. Currently, no definite treatment policy is available and only empirical treatment is applied. This case is an important for the diagnosis and treatment of this bacterial infection on nasopharynx.

Just Seeing Is Not Enough for Believing: Immunolabelling as Indisputable Proof of SARS-CoV-2 Virions in Infected Tissue

Background: There is increasing evidence that identification of SARS-CoV-2 virions by transmission electron microscopy could be misleading due to the similar morphology of virions and ubiquitous cell structures. This study thus aimed to establish methods for indisputable proof of the presence of SARS-CoV-2 virions in the observed tissue. Methods: We developed a variant of the correlative microscopy approach for SARS-CoV-2 protein identification using immunohistochemical labelling of SARS-CoV-2 proteins on light and electron microscopy levels. We also performed immunogold labelling of SARS-CoV-2 virions. Results: Immunohistochemistry (IHC) of SARS-CoV-2 nucleocapsid proteins and subsequent correlative microscopy undoubtedly proved the presence of SARS-CoV-2 virions in the analysed human nasopharyngeal tissue. The presence of SARS-CoV-2 virions was also confirmed by immunogold labelling for the first time. Conclusions: Immunoelectron microscopy is the most reliable method for distinguishing intracellular viral particles from normal cell structures of similar morphology and size as virions. Furthermore, we developed a variant of correlative microscopy that allows pathologists to check the results of IHC performed first on routinely used paraffin-embedded samples, followed by semithin, and finally by ultrathin sections. Both methodological approaches indisputably proved the presence of SARS-CoV-2 virions in cells.

WNT8B as an Independent Prognostic Marker for Nasopharyngeal Carcinoma

Background: Members of the Wnt signaling pathway have been shown to play a role in nasopharyngeal carcinoma (NPC) progression. Aim: The purpose of this study was to investigate WNT8B protein expression in NPC patients using tissue microarray (TMA) analysis and to evaluate its correlation with patient survival and clinical parameters. Methods: A total of 82 NPC cases, together with six normal nasopharyngeal tissue samples, were targeted to construct the TMA blocks. The WNT8B protein expression was evaluated by immunohistochemistry and its correlation to the clinicopathological features was investigated. Results: Sixty-two of 82 (75.6%) cases exhibited high WNT8B protein expression while 20/82 (24.4%) cases appeared to have low WNT8B expression. The univariate analysis revealed that systemic metastasis was associated with patient 5-year survival. The multivariate Cox proportional hazard regression analysis showed that WNT8B expression and systemic metastasis were significantly associated with the survival of NPC patients. Furthermore, there was no correlation found between the WNT8B protein expression and other clinicopathological parameters. Conclusion: Our results suggest that the expression of WNT8B is associated with NPC patients’ survival and could serve as an independent prognostic factor for NPC patients.

Alteration of L-Dopa decarboxylase expression in SARS-CoV-2 infection and its association with the interferon-inducible ACE2 isoform

L-Dopa decarboxylase (DDC) is the most significantly co-expressed gene with ACE2, which encodes for the SARS-CoV-2 receptor angiotensin-converting enzyme 2 and the interferon-inducible truncated isoform dACE2. Our group previously showed the importance of DDC in viral infections. We hereby aimed to investigate DDC expression in COVID-19 patients and cultured SARS-CoV-2-infected cells, also in association with ACE2 and dACE2. We concurrently evaluated the expression of the viral infection- and interferon-stimulated gene ISG56 and the immune-modulatory, hypoxia-regulated gene EPO. Viral load and mRNA levels of DDC, ACE2, dACE2, ISG56 and EPO were quantified by RT-qPCR in nasopharyngeal swab samples from COVID-19 patients, showing no or mild symptoms, and from non-infected individuals. Samples from influenza-infected patients were analyzed in comparison. SARS-CoV-2-mediated effects in host gene expression were validated in cultured virus-permissive epithelial cells. We found substantially higher gene expression of DDC in COVID-19 patients (7.6-fold; p = 1.2e-13) but not in influenza-infected ones, compared to non-infected subjects. dACE2 was more elevated (2.9-fold; p = 1.02e-16) than ACE2 (1.7-fold; p = 0.0005) in SARS-CoV-2-infected individuals. ISG56 (2.5-fold; p = 3.01e-6) and EPO (2.6-fold; p = 2.1e-13) were also increased. Detected differences were not attributed to enrichment of specific cell populations in nasopharyngeal tissue. While SARS-CoV-2 virus load was positively associated with ACE2 expression (r≥0.8, p<0.001), it negatively correlated with DDC, dACE2 (r≤−0.7, p<0.001) and EPO (r≤−0.5, p<0.05). Moreover, a statistically significant correlation between DDC and dACE2 expression was observed in nasopharyngeal swab and whole blood samples of both COVID-19 and non-infected individuals (r≥0.7). In VeroE6 cells, SARS-CoV-2 negatively affected DDC, ACE2, dACE2 and EPO mRNA levels, and induced cell death, while ISG56 was enhanced at early hours post-infection. Thus, the regulation of DDC, dACE2 and EPO expression in the SARS-CoV-2-infected nasopharyngeal tissue is possibly related with an orchestrated antiviral response of the infected host as the virus suppresses these genes to favor its propagation.

Microdebrider: a painless and effective technique for adenoidectomy; comparative study with curette assisted adenoidectomy

<p><strong>Background: </strong>Adenoid is a nasopharyngeal tissue which forms Waldeyer's ring as said by Meyer, 1968. Adenoids become demonstrable with signs of CSOM with adenoid hyperplasia, recurrent rhino-sinusitis, characteristic ‘adenoid facies’, nasal obstruction, mouth breathing, snoring, drooling of saliva and speech abnormalities and dental malocclusion. Adenoidectomyis the common surgery done using various techniques like curette, microdebrider and many more. In this study we evaluate and compare the efficacy of adenoidectomy by microdebrider verses curette assisted adenoidectomy. Aim of the study was to evaluate and compare the efficacy and benefits of adenoidectomy by microdebrider with curette assisted adenoidectomy.</p><p><strong>Methods: </strong>This is a prospective randomized single-blind study done for 1 year. Total 150 patients were included which were diagnosed as adenoid hypertrophy based on clinical and radiological examination and were equally divided in 2 group for both the procedures (curette and microdebrider).</p><p><strong>Results: </strong>Patients show good response to the treatment with microdebrider assisted adenoidectomy with less complication and early recovery.</p><p><strong>Conclusions: </strong>We observe that proper examination and early surgical intervention using modern technique i.e., microdebrider reduces the time, residual tissue with less complication and promote early recovery.</p>

A first case report of nasopharyngeal mycobacterium abscessus ssp. massilliense infection

BackgroundMycobacterium abscessus ssp. massiliense is a non-tuberculous mycobacteriosis and was subdivided from Mycobacterium abscessus in 2006. There are no reports to date on nasopharyngitis caused by Mycobacterium abscessus ssp. massiliense.Case PresentationA 45-year old woman was referred to Yokohama City University Medical Center with an 18-month history of recurrent nasopharyngitis and presented with pain in the throat. Mycobacterial tissue culture and polymerase chain reaction testing revealed the presence of Mycobacterium abscessus ssp. massiliense in the nasopharyngeal tissue. This patient underwent surgery, followed by multiple rounds of chemotherapy with oral and intravenous antibiotic agents for four months. She has had no recurrence during the 8 months since treatment.ConclusionThere are few reports on Mycobacterium abscessus ssp. massiliense infection in the head and neck region, and none in the pharynx. To our knowledge, this is the first report of a patient with a nasopharyngeal Mycobacterium abscessus ssp. massiliense infection. It is difficult to prove the presence of Mycobacterium abscessus ssp. massiliense in a pharyngeal “swab,” and tissue culture from a biopsy specimen is mandatory for the identification of the species. Currently, no definite treatment policy is available and only empirical treatment is applied. Further reports are needed to accumulate supporting evidence.

Identification of Key Pathways and Genes in Nasopharyngeal Carcinoma Based on Bioinformatics Analysis

Purpose: The purpose of this study is to identify novel molecular markers and potential molecular targets for NPC based on bioinformatics analysis.Methods: We used bioinformatics to analyze one miRNA and two mRNA expression microarray datasets from the Gene Expression Omnibus database. The study included nasopharyngeal tissue samples from 57 patients with NPC and 32 patients without NPC. Fifty-one screened differentially expressed genes (DEGs) were evaluated by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) signal pathway enrichment analyses, and a protein-protein interaction (PPI) network was constructed. Results: The GO analysis results showed that the DEGs were mainly related to cell cycle checkpoints, cell division, and DNA synthesis during DNA repair. The KEGG analysis results suggested that the DEGs were mainly associated with extracellular matrix receptor interactions. In the PPI network, we identified RAD51AP1, MAD2L1, SPP1, CCNE2, CNTNAP2, and MELK as hub genes, clustered a key module, and identified eight key transcription factors: TFII-I, Pax-5, STAT4, GR-alpha, YY1, C/EBPβ, GRβ, and TFIID. Conclusion: The hub genes and signaling pathways identified above may play an important role in NPC development and provide ideas for the selection of valuable prognostic markers and the development of new molecular-targeted drugs.

Korelasi ekspresi IL-6 dengan STAT3 pada metastasis penderita KNF WHO tipe 3 LMP-1 positif

Latar belakang: Karsinogenesis karsinoma nasofaring sangat kompleks, dan disebabkan oleh interaksi antara infeksi kronis Epstein-Barr Virus (EBV), faktor lingkungan, dan perubahan genetik serta epigenetik. Latent Membrane Protein (LMP)-1 merupakan antigen utama EBV. LMP-1 diyakini menstimulasi ekspresi sitokin yang memengaruhi perilaku sel epitel, salah satunya adalah Interleukin (IL)- 6. IL-6 akan mengaktivasi jalur Signal Transducer and Activator of Transcription (STAT)3. Peningkatan ekspresi IL-6 dan STAT3 memiliki kaitan erat dalam lingkungan mikro tumor KNF, namun peran IL-6 dan STAT3 dalam modulasi migrasi dan invasi sel KNF masih belum jelas diketahui. Tujuan: Mengetahui korelasi antara ekspresi IL-6 dan ekspresi STAT3 di jaringan nasofaring dengan metastasis penderita KNF WHO tipe III LMP-1 positif. Metode: Penelitian observasional analitik dengan pendekatan cross-sectional yang melibatkan 15 penderita KNF WHO tipe III LMP-1 positif. Pemeriksaan ekspresi LMP-1, IL-6, dan STAT3 menggunakan metode pewarnaan imunohistokimia, dan hasilnya dihitung dengan menggunakan software ImmunoRatio. Penentuan stadium klinis metastasis berdasarkan pemeriksaan klinis dan penunjang radiologis, kemudian dievaluasi nilai TNM menggunakan AJCC 2017. Hasil: Analisis statistik ekspresi IL-6 dan STAT3 menunjukkan korelasi yang signifikan (p=0,020) dengan koefisien korelasi r=0,592. Tidak terdapat perbedaan ekspresi IL-6 yang bermakna di antara penderita stadium III, IVa, dan IVb (p=0,116). Terdapat perbedaan ekspresi STAT3 yang signifikan di antara penderita dengan stadium III, IVa, dan IVb (p=0,038). Kesimpulan: Semakin tinggi ekspresi IL-6 maka ekspresi STAT3 pada jaringan nasofaring penderita KNF WHO tipe III LMP-1 positif semakin meningkat. Semakin tinggi stadium klinis, maka ekspresi STAT3 pada jaringan nasofaring penderita KNF WHO tipe III LMP-1 positif akan meningkat. Kata kunci: karsinoma nasofaring, Virus Epstein-Barr, LMP-1, IL-6, STAT3 ABSTRACT Background:Carcinogenesis of NPC is complex interactions among chronic EBV infection, environmental factors, genetic and epigenetic. LMP-1 is EBV’s antigen most expressed in NPC cases. LMP-1 is believed to stimulate expression of cytokines that affect the behavior of epithelial cells, i.e. Interleukin-6 (IL-6) which will activate the STAT3 pathway. Increased expression of IL-6 and STAT3 has a close relationship in tumor microenvironment of NPC patients, but the role of IL-6 and STAT3 themselves in modulation of migration and invasion of NPC cells are not yet fully understood. Purpose: To find out the correlation between IL-6 expression and STAT3 expression in the nasopharyngeal tissue of NPC patients with LMP-1 positive metastatic WHO type III. Method: Observational analytic study with cross-sectional approach involving 15 patients WHO type III LMP-1 positive NPC patients. Examination of LMP-1, IL-6, and STAT3 using immunohistochemical, and the results were calculated using ImmunoRatio. Clinical staging of metastases based on clinical examination and radiological imaging then synchronized with AJCC 2017. Result: Expressions of IL-6 and STAT3 showed significant correlation (p=0.020) with coefficient r=0.592. There were no differences in IL-6 expression among patients with stage III, IVa, and IVb (p=0.116). There were significant differences in STAT3 expression among patients with stage III, IVa, and IVb (p=0.038). Conclusion: There was a significant positive correlation between IL-6 and STAT3 in tissue from WHO type III LMP-1 positive NPC patients. The higher the clinical stage, the expression of STAT3 in the nasopharyngeal tissue of positive WHO type III LMP-1 NPC patients will increase.

Pengaruh ekspresi p53 dan HIF1 terhadap peningkatan laktat jaringan nasofaring pada pasien karsinoma nasofaring

Latar Belakang: Karsinoma nasofaring (KNF) merupakan keganasan tersering pada kepala dan leher. Pilihan terapi KNF adalah radioterapi dan kemoterapi yang berhubungan dengan toksisitas, resistensi obat, dan rekurensi. Intervensi metabolik yang didasarkan pada perubahan metabolisme sel kanker merupakan salah satu strategi terapi kanker pada saat ini. Untuk dapat mengetahuinya perlu dipahami pengaruh ekspresi p53 dan hypoxia-inducible factor 1 (HIF1) terhadap peningkatan kadar laktat jaringan nasofaring pada pasien KNF. Tujuan: Mengetahui pengaruh ekspresi p53 dan HIF1 terhadap peningkatan kadar laktat jaringan nasofaring, dan untuk mengetahui kesesuaian antara kadar laktat darah dengan laktat jaringan nasofaring. Metode: Penelitian cross sectional melibatkan 10 subjek, dilakukan biopsi nasofaring dengan tuntunan nasoendoskopi untuk pemeriksaan histopatologi, ekspresi p53 dan HIF1 dengan imunohistokimia, laktat jaringan nasofaring dengan colorimetric, dan laktat darah. Hasil: Seluruh subjek mengalami peningkatan ekspresi p53 dan HIF1 dengan rerata p53 19,53±7,37 dan HIF1 24,30±12,28. Seluruh subjek penelitian memiliki kadar laktat jaringan meningkat, dengan rerata kadar laktat 0,67±0,39. Kadar laktat darah subjek cenderung meningkat dengan rerata 2,93±0,65. Terdapat pengaruh peningkatan ekspresi p53 terhadap peningkatan kadar laktat jaringan (p=0,002). Terdapat pengaruh peningkatan ekspresi HIF1 terhadap peningkatan kadar laktat jaringan (p=0,042). Tidak terdapat kesesuaian antara kadar laktat darah dengan laktat jaringan nasofaring (p=0,000). Kesimpulan: Peningkatan ekspresi p53 dan HIF1 berpengaruh terhadap peningkatan kadar laktat jaringan nasofaring pada pasien KNF, namun kadar laktat darah tidak menggambarkan kadar laktat jaringan nasofaring. ABSTRACTBackground: Nasopharyngeal carcinoma (NPC) is the most frequent malignancy of the head and neck. The options of NPC therapy are radiotherapy and chemotherapy, associated with toxicity, drug resistance, and recurrence. Metabolic intervention based on changes in cancer cell metabolism is currently one of the strategies of cancer therapy. Aim: To determine the impact of p53 and hypoxia-inducible factor 1 (HIF1) expression on elevated lactate levels of nasopharyngeal tissue, and to determine the compatibility between blood lactate and nasopharyngeal tissue lactate levels in patients with NPC. Method: This cross-sectional study involved 10 subjects who underwent nasopharyngeal biopsy for histopathologic examination, p53 and HIF1 expression using immunohistochemistry, lactate of nasopharyngeal tissue using colorimetric, and blood lactate. Results: All subjects had increased expression of p53 and HIF1 with p53 mean of 19.53±7.37 and HIF1 mean of 24.30±12.28. All subjects had elevated tissue lactate levels, with lactate levels mean of 0.67±0.39. The blood lactate level of the subjects increased, with blood lactate level mean of 2.93±0.65. There was a significant increasing impact of p53 expression on tissue lactate elevated level (p=0.002) and a significant increasing impact of HIF1 expression on tissue lactate elevated level (p=0.042). There was no correlation between lactate levels of blood lactate and nasopharyngeal tissue (p=0.000). Conclusion: Increased expression of p53 and HIF1 had an effect on increased levels of lactate nasopharyngeal tissue in NPC patients, but blood lactate levels did not have a correlation with lactate levels of nasopharyngeal tissue.