Detection Of Epstein-Barr Virus Research Articles

Molecular detection of Epstein-Barr virus in paraffin-embedded tissue samples of patients suffering gastric cancer in Ahvaz, Iran: a case-control study

Background and Objectives: Gastric cancer (GC) is the third most common cause of cancer-related mortality. Epstein-Barr virus (EBV) is associated with several human tumors. The present research was performed to investigate the prevalence of EBV-associated gastric cancer (EBVaGC) among Iranian patients. Materials and Methods: Seventy cases of gastric cancer and 30 cases of gastric ulcer, all preserved in formalin-fixed paraf- fin-embedded (FFPE), were examined in a case-control study conducted between 2011 and 2018. The specimens underwent analysis to detect the presence of the EBV genome using a Nested-PCR method targeting EBNA1. Subsequently, samples testing positive for the EBNA1 underwent further testing for the presence of the EBER gene using PCR. Finally, Positive samples were subjected to sequencing. Results: Five out of 70 cases (7%) were found to be positive for EBV based on EBNA1 testing, while all EBNA1 positive samples were negative for EBER. Notably, EBV was not detected in patients with gastric ulcer. The mean age of EBV-posi- tive gastric carcinomas pateints was 64.5 years. Within this group, 60% were male and 40% were female. A higher prevalence of EBV association was observed in diffuse-type cases, with 60% (3 out of 24) testing positive, compared to intestinal-type cases where 40% (2 out of 46) were EBV-positive. Most cases of EBVaGC belonged to grade Ⅰ. Conclusion: This research demonstrates a low prevalence of EBVaGC in Iran. Discrepancies in EBVaGC occurrence among countries could be attributed to epidemiological variables and dietary practices. A comprehensive studies will provide signif- icant contributions to understanding of its etiology

APOBR Is Downregulated in EBV+ Tonsils of Children with Obstructive Sleep-Disordered Breathing.

Background: Obstructive sleep-disordered breathing (oSDB) is a heterogeneous phenotype that is increasing in prevalence worldwide and has many potential comorbidities that could severely affect quality of life. There is a need to identify biomarkers for oSDB and its comorbidities to improve clinical management, particularly in children. Methods: We performed bulk mRNA-sequencing, differential expression analysis, and qPCR replication of selected differentially expressed genes (DEGs) using RNA samples extracted from tonsils of children with oSDB. Two variables were used as classifier, namely, detection of Epstein-Barr virus (EBV) in tonsils and need for continuous positive airway pressure (CPAP) treatment. Standard statistical tests were used to determine associations across clinical, EBV, and DEG variables. Results: Nineteen genes were dysregulated in tonsils that are EBV+ or from children needing CPAP. Of these genes, APOBR was downregulated in both EBV+ and CPAP+ tonsils, and this downregulation was replicated by qPCR in an independent set of pediatric samples. In the tonsils of adult patients with oSDB, APOBR was positively correlated with age, and potentially with diastolic blood pressure. Conclusions: Taken together, APOBR and DEGs in tonsillar tissues may be useful as potential biomarkers of oSDB severity and comorbidity across the lifespan, with APOBR levels being dependent on latent EBV infection.

Exploring atypical manifestations and multisystem involvement of Epstein-Barr virus infection in hospitalized pediatric patients from Mexico: insights from a tertiary hospital (2012-2022).

Epstein-Barr virus (EBV) infection, with a global prevalence exceeding 95%, typically manifests in children as infectious mononucleosis. However, clinical practice frequently encounters diverse atypical presentations characterized by multisystem involvement, often resulting in an unfavorable clinical course. Our objective is to describe the clinical manifestations and results of EBV infection in a tertiary pediatric hospital in Mexico. An observational, transversal, retrospective, and descriptive study that included a systematic review of medical records (2012-2022) of patients under 18 years of age with detectable EBV particles in peripheral blood. The study included 26 patients with a median age of 5 years and a male predominance of 53.8%. Predominant symptoms were fever (85%) and lymphadenopathy (35%). Sixty-five percent had severe and atypical manifestations, including pneumonia and hepatic, hematologic-oncologic, and autoimmune diseases. Anemia, thrombocytopenia and leukopenia were common, with lymphocytosis in 19% of cases. The median EBV viral load was 2816 copies/mL (range: 555-355,500 copies/mL). Four deaths related to EBV infection were reported. Viral load in these cases also varied widely from 594 to 121,000 copies/mL. Supportive care was administered to 85% of patients, while others received antiviral treatment, steroids, and rituximab. Atypical manifestations were common, especially in children with multisystem involvement. EBV should be considered as a potential contributor to a diverse spectrum of clinical presentations, emphasizing the need for comprehensive evaluation and awareness in clinical diagnosis.

Disparities in the Levels of Whole-Blood Epstein-Barr Virus between the Cancer and Non-Cancer Populations in Zhejiang, China.

This study aimed to investigate the prevalence of Epstein-Barr virus (EBV) infection in patients with and without cancer. A total of 26,648 participants who underwent whole-blood EBV DNA (WBEBV) assays between January 1, 2020, and August 31, 2023, were included. The chi-square test was used for categorical data analysis, and R software was used to analyze the differences in EBV DNA load levels and the diagnostic capabilities of WBEBV. Positive rates were 10.2% and 25.4% for healthy controls (HC) and patients, respectively. The positivity rate for EBV-associated neoplasms (EN) was the highest at 7.53%, followed by leukemia (Le) at 5.49%. The subgroup analysis showed that the positivity rate for abnormal proliferation or hyperplasia (APH) was 31.9%, followed by 30.5% for Le. The WBEBV of patients with transplants (TP), especially living-related transplants (LT), was the highest among all subgroups. WBEBV at diagnosis was used to differentiate between infectious mononucleosis (IM) and chronic active Epstein-Barr virus (CAEBV), with a sensitivity of 67.4% (95% confidence interval [ CI]: 57.6-75.8) and specificity of 72% (95% CI: 63.3-79.3). We conclude that the prevalence of EBV infection is low in the healthy population in this region and that a high EBV load at baseline is more common in LT, IM, and Lymphocyte Leukemia (LL). This study used a large-sample survey to characterize the prevalence of whole-blood EBV levels in various diseases, including the stages and subtypes. The EBV detection rate was higher in patients with malignant disease than in those with benign disease. Our study provides clinicians with baseline information regarding EBV-associated diseases.

Unveiling the Impact of Epstein-Barr Virus on the Risk of Prostate Cancer: A Mendelian Randomization Study

Given the consistent detection of Epstein-Barr virus (EBV) in prostate tissues and the clinical evidence suggesting its involvement in prostate cancer (PCa), the potential association between EBV infection and PCa warrants further investigation. This study aimed to assess the causal relationship between EBV infection and PCa using Mendelian randomization (MR). We utilized data from a publicly available genome-wide association study (GWAS) on PCa, alongside data on five serum anti-EBV virus-related antibodies. Our findings indicate a potential causal link between serum EBV EA-D antibody levels and an increased risk of PCa. These results highlight the need for additional research to elucidate the mechanisms by which EBV may contribute to the progression of PCa, potentially offering new insights into its pathogenesis and therapeutic targets.

Association Between Antibodies That Bind Epstein-Barr Virus (EBV) gp350 and gH/gL and Shedding of EBV in Saliva From Nasopharyngeal Carcinoma Multiplex Family Members in Taiwan.

Elevated levels of Epstein-Barr virus (EBV) gp350 and gH/gL antibodies have been associated with a lower risk of developing nasopharyngeal carcinoma (NPC), although the evidence remains inconclusive and unexplained. We conducted a longitudinal study within a high-risk Taiwanese cohort, analyzing total immunoglobulin against EBV-gp350 and -gH/gL in blood and EBV DNA shedding in saliva. Contrary to our hypothesis-that elevated levels of antibodies previously shown to be associated with a lower NPC risk should result in a decrease in EBV shedding in saliva-higher anti-gp350 antibodies at baseline were significantly associated with detectable EBV DNA in saliva at follow-up (odds ratio [OR], 1.99 [95% confidence interval {CI}, 1.03-3.97]; P = .04). Higher anti-EBV-gH/gL antibodies at baseline were not significantly associated with risk of detectable EBV DNA at follow-up (OR, 0.69 [95% CI, .35-1.32]; P = .26). These findings underscore the complexity of virus-host interactions and emphasize the need for further investigations into their role in EBV-associated diseases.

Longitudinal tracing of on-treatment plasma Epstein-Barr virus DNA as a biomarker for real-time dynamic risk monitoring in patients with nasopharyngeal carcinoma: The EP-SEASON study.

136 Background: Plasma Epstein-Barr virus (EBV) DNA is a biomarker for nasopharyngeal carcinoma (NPC). However, whether longitudinal on-treatment EBV DNA tracing would inform real-time recurrence risk in NPC remains to be elucidated. To address this issue, we conducted EP-SEASON (NCT03855020), a prospective cohort study of longitudinal on-treatment EBV DNA in EBV-positive NPC patients. Methods: Non-metastatic NPC with detectable pretreatment EBV DNA that received standard sequential chemo-radiotherapy were recruited. Serial EBV DNA in blood specimens were collected across pretreatment, 3-week after each induction chemotherapy (IC), every-week during radiotherapy (RT), within 1-week and 1-3 months upon RT completion. EBV DNA extracted from plasma was quantified by a real-time quantitative polymerase chain reaction assay targeting the BamHI-W region of the EBV genome. Longitudinal changing patterns of EBV DNA and their clinical relevance were analyzed. Results: Between May 2019 and April 2021, a total of 1000 patients underwent per-protocol EBV DNA assessments and treatment; 248 and 752 patients received concurrent chemoradiotherapy (CCRT, subcochortno-IC) and IC plus CCRT (subcochortIC), respectively. Longitudinal changes and clearance rates of EBV DNA displayed unique patterns during IC and RT phases. Throughout IC and RT phases, the rate of EBV DNA clearance decreased with repeated IC cycles; interestingly, the rate re-rose upon the introduction of RT, and then dropped to low levels (<10%/week) after the 4th week of RT (post-RT4w). Patients in subcochortno-IC and subcochortIC also exhibited differential clearance patterns during RT, with the rate of clearance being higher in subcochortno-IC before the 2nd week of RT (post-RT2w). Notably, despite the prognostic significance of EBV DNA at each timepoint ( Pall < 0.05), longitudinal EBV DNA displayed a linkage changing map with dynamic recurrence risks: 3-year disease-free survival (DFS) rate steadily dropped with delayed EBV DNA clearance during IC; it re-rose at the 2nd RT week upon EBV DNA clearance, followed by a sharp decrease at the 4th RT week in subcochortIC. In contrast, 3-year DFS rate continuously decreased with delayed EBV DNA clearance in subcochortno-IC. Finally, we identified different phenotypes of whole-course circulating tumor DNA changing dynamics that showed distinct prognosis ( P < 0.05). Conclusions: Longitudinal on-treatment EBV DNA tracing could forecast real-time risks and facilitate risk-adapted decision-making in NPC.

Quantitatively assessed blood leukocyte Epstein–Barr virus in adult HIV-infected patients

Coinfection with the human immunodeficiency virus (HIV) and Epstein–Barr virus (EBV) represents a current biomedical problem. The purpose of the study was to evaluate blood leukocyte EBV detection rate and viral load in adult HIV-infected patients. Materials and methods. There were examined blood leukocytes collected from 138 HIV(+) and 68 HIV(–) individuals aged 20–69 years. Statistical analysis was carried out differentiated according to the stages of HIV infection, the CD4+ T-lymphocyte count, and adherence to antiretroviral therapy. Results. It was shown that EBV DNA was detected significantly more often in HIV(+) vs HIV(–) individuals (70.3±3.9% and 48.5±6.1%, p = 0.008), with EBV viral load comprising 18 [5; 139] versus 2 [1; 3] copies/105 cells (р 0.001), respectively. It has been shown that the group of HIV(+) patients is heterogeneous in the frequency of EBV detection and viral load, with the peak EBV frequency (86.7±6.2%) and DNA (121 [34; 252] copies/105 cells) level observed in “naive” patients with severe immunodeficiency. Among “experienced” patients receiving therapy, the relative risk of detecting EBV DNA with low treatment adherence was significantly higher compared to those who developed high adherence (р 0.05). When the HIV viral load reached undetectable level, EBV DNA concentration was significantly lower where HIV RNA was detectable (1 [0; 8] versus 15 [1; 162] copies/105 cells, р 0.001). Detection of EBV DNA is associated with higher HIV viral load level and lower CD4+ T-lymphocyte count compared to patients with undetected EBV DNA. A relationship has been established between the CD4+ T-lymphocyte count in HIV(+) patients and the likelihood of active EBV infection. A threshold cut-off of 200 cells/μl was determined. CD4+ T-lymphocyte count 200 cells/μl vs ≥ 200 cells/μl (р 0.001) is associated with a 3.3-fold higher risk of detecting active EBV infection. Conclusion. It is necessary to continue interdisciplinary research to improve early diagnostics of EBV-associated diseases in HIV-infected individuals.

The efficacy of adjuvant chemotherapy in patients with different midpoint-radiotherapy Epstein-Barr virus DNA plasma loads

ObjectivesThis study aimed to evaluate the efficacy of adjuvant chemotherapy (AC) in patients with different midpoint-radiotherapy (mid-RT) Epstein-Barr virus (EBV) DNA plasma loads for locoregionally advanced nasopharyngeal carcinoma (NPC), and to provide decision-making regarding the use of AC. Materials and methodsA total of 675 consecutive patients diagnosed with stage III–IVa NPC were enrolled in this study. All patients underwent concurrent chemoradiotherapy (CCRT), either with or without induction chemotherapy or AC, or a combination of both. The primary endpoint of this study was progression-free survival (PFS). ResultsAmong the 675 enrolled patients, 248 (36.7 %) received AC and 427 (63.3 %) were only observed after CCRT. In total, 149 (22.1 %) patients had detectable mid-RT EBV DNA levels, whereas 526 (77.9 %) had undetectable mid-RT EBV DNA levels. Patients with detectable mid-RT EBV DNA had worse 5-year PFS than those with undetectable mid-RT EBV DNA (74.8 % vs. 81.9 %, P = 0.045). AC group showed significantly better 5-year PFS than observation in patients with detectable mid-RT EBV DNA (82.8 % vs. 66.8 %; HR, 0.480; 95 % CI 0.250–0.919, P = 0.027). Multivariate analyses demonstrated that the treatment methods (AC vs. observation) were independent prognostic factors for PFS (HR, 0.37; 95 % CI 0.19–0.74, P = 0.005). However, in patients with undetectable mid-RT EBV DNA (5-year PFS: HR 0.873, 95 % CI 0.565–1.349, P = 0.52), AC group showed no survival benefit for observation. ConclusionAC could reduce the risk of disease progression compared to observation in patients with detectable mid-RT EBV DNA. Our findings suggest that AC is effective in patients at a high risk of treatment failure.

Presence of herpesviruses, parvoviruses, and polyomaviruses in sinonasal lymphoma

PurposeSinonasal lymphoma (SL) is a rare lymphatic neoplasm of the nasal cavities, paranasal sinuses and nasopharynx. Whereas some risk factors for SL subtypes have been identified, their aetiology is unknown. Along with other predisposing factors, the viral association of lymphomas, such as Epstein-Barr virus (EBV) and Burkitt and Hodgkin lymphomas, is well-established. Modern molecular biology techniques have enabled the discovery of novel human viruses, exemplified by the protoparvovirus cutavirus (CuV), associated with cutaneous T-cell lymphoma. These findings, and the anatomical location of the sinonasal tract with its rich microbiome and infectious agents, justify in-depth studies among SL.MethodsWe analysed the presence of 20 viruses of Orthoherpesviridae, Parvoviridae, and Polyomaviridae by qPCR in 24 SL tumours. We performed RNAscope in situ hybridisation (RISH) to localize the viruses. Parvovirus-specific IgG was analysed by enzyme immunoassay and targeted next-generation sequencing (NGS) was applied to detect CuV in plasma.ResultsWe detected viral DNA in 15/24 (63%) tumours; nine of EBV, six of human herpesvirus (HHV) -7, four each of HHV-6B and parvovirus B19, two of cytomegalovirus, and one each of CuV and Merkel-cell polyomavirus. We found tumours with up to four viruses per tumour, and localized CuV and EBV DNAs by RISH. Two of the ten plasma samples exhibited CuV IgG, and one plasma sample demonstrated CuV viremia by NGS.ConclusionViruses were frequent findings in SL. The EBV detection rate was high in diffuse large B-cell lymphoma, and co-detections with other viruses were prevalent.

Human papillomavirus and Epstein-Barr virus infection in benign thyroid lesions.

The objective was to investigate the correlation between Epstein-Barr virus (EBV) and human papillomavirus (HPV) infection in the development of benign thyroid lesions. 29 cases of Hashimoto's thyroiditis (HT), 133 cases of thyroid adenoma, and 34 cases of HT with thyroid adenoma paraffin embedded tissue samples were used for EBV and HPV quantitative detection. None of the tissue samples carried HPV DNA. In HT tissue samples, the positive rate of EBV was 55.2% (16/29). In thyroid adenoma tissue samples, the positive rate was 37.6% (50/133). In HT combined with thyroid adenoma tissue samples, the positive rate of EBV was 67.6% (23/34). There was no correlation between EBV infection and clinical features such as age and gender. The occurrence and development of benign thyroid lesions are closely related to EBV infection. HT combined with thyroid adenoma may be more susceptible to EBV infection than simple HT and thyroid adenoma, which provides a new idea for the diagnosis and treatment of benign thyroid lesions.

A Multiplex Fluorescence of Loop Primer Upon Self-Dequenching Loop-Mediated Isothermal Amplification Assay for the Detection of Epstein-Barr Virus and Human Parvovirus B19 in Clinical Transplant Samples.

Viral infections are major causes of mortality in solid-organ and hematopoietic stem cell transplant recipients. Epstein-Barr virus (EBV) and Parvovirus B19 (B19V) are among the common viral infections after transplantation and were recommended for increased screening in relevant guidelines. Therefore, the development of rapid, specific, and cost-effective diagnostic methods for EBV and B19V is of paramount importance. We applied Fluorescence of Loop Primer Upon Self-Dequenching Loop-mediated Isothermal Amplification (FLOS-LAMP) for the first time to develop a novel multiplex assay for the detection of EBV and B19V; the fluorophore attached to the probe are self-quenched in unbound state. After binding to the dumbbell-shaped DNA target, the fluorophore is dequenched, resulting in fluorescence development. The novel multiplex FLOS-LAMP assay was optimized by testing various ratios of primer sets. This novel assay, with great specificity, did not cross-react with the common virus. For the detection of EBV and B19V, the limits of detection could reach 969 and 798 copies/μL, respectively, and the assay could be completed within 25 min. Applying this novel assay to detect 200 clinical transplant individuals indicated that the novel assay had high specificity and good sensitivity. We developed multiplex FLOS-LAMP assay for the detection of EBV and B19V, which has the potential to become an important tool for clinical transplant patient screening.

Herpesviridae and Atypical Bacteria Co-Detections in Lower Respiratory Tract Samples of SARS-CoV-2-Positive Patients Admitted to an Intensive Care Unit.

Shortly after the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), cases of viral, bacterial, and fungal coinfections in hospitalized patients became evident. This retrospective study investigates the prevalence of multiple pathogen co-detections in 1472 lower respiratory tract (LRT) samples from 229 SARS-CoV-2-positive patients treated in the largest intensive care unit (ICU) in Slovenia. In addition to SARS-CoV-2, (rt)RT-PCR tests were used to detect cytomegalovirus (CMV), Epstein-Barr virus (EBV), herpes simplex virus 1 (HSV-1), herpes simplex virus 2 (HSV-2), varicella zoster virus (VZV), and atypical bacteria: Chlamydia pneumoniae, Mycoplasma pneumoniae and Legionella pneumophila/spp. At least one co-detection was observed in 89.1% of patients. EBV, HSV-1, and CMV were the most common, with 74.7%, 58.1%, and 38.0% of positive patients, respectively. The median detection time of EBV, HSV-1, and CMV after initial SARS-CoV-2 confirmation was 11 to 20 days. Bronchoalveolar lavage (BAL) and tracheal aspirate (TA) samples showed equivalent performance for the detection of EBV, CMV, and HSV-1 in patients with both available samples. Our results indicate that SARS-CoV-2 infection could be a risk factor for latent herpesvirus reactivation, especially HSV-1, EBV, and CMV. However, additional studies are needed to elucidate the clinical importance of these findings.

Detection of Epstein-Barr virus among chronic kidney disease patients in Najaf, Iraq

Abstract Many people across the world have been infected with Epstein Barr virus (EBV), a kind of a human herpes virus. Loss of kidney function happens progressively with time in those with chronic kidney disease (CKD). Individuals with CKD may be more susceptible to viral infections such as EBV due to their compromised immune systems. The purpose of this research is to quantify the frequency with which EBV infection occurs in patients with CKD in the Al-Najaf governorate. Blood samples from 50 people with CKD and 50 healthy controls were collected for the study at the Specialized Centre for Diseases and Kidney Transplant at Al-Sadder Medical City in Al-Najaf city. Nineteen of the CKD patients were men, whereas 31 were women. The age range of individuals who took part in the research varied from 17 to 47. The time frame for this study’s data gathering was from January 2022 to December 2022. Fifty hemodialysis patients gave blood samples for analysis. Researchers measured anti-EBV IgG antibodies using the ELISA method to evaluate the efficacy of humoral immunity. Eleven (22% of all samples) were found to have positive results for Epstein Barr nuclear antigen2 (EBNA2) IgG. This number was 43.1% of all males and 38.4% of all females. However, the serum concentration was found to be statistically significantly higher in the age range of 17-26 years (p0.05) than in any other age group. The RT-qPCR method was also applied to detect EBV infection in the study. Only 18 (36% of the sample) of the 50 patients had positive RT-qPCR results for EBV. This number was 15.7% of all males and 48.4% of all females; however, the positivity rate increased to 71.4% in the (17-26) age group and decreased to 28.5% in those older than 57. Results from the control group were negative for EBNA2 IgG antibodies and EBV DNA. Findings of this research demonstrate that the prevalence of EBV antibodies is considerably higher among patients undergoing hemodialysis than in the group serving as a control, regardless of age. In addition, the results of the study support the use of real-time PCR for detecting EBV among patients with CKD, due to its high sensitivity and specificity.

Epstein-Barr virus (EBV) DNAemia in saliva of viral encephalitis and nasopharyngeal carcinoma patients.

Epstein-Barr virus (EBV) is a lymphotropic virus that causes diseases ranging from a flu-like illness called infectious mononucleosis to nasopharyngeal carcinoma, Burkitt's lymphoma, and central nervous system (CNS) infection. Detection of EBV DNA is usually done using whole blood samples taken from the patients. We undertook the detection of EBV in blood, cerebrospinal fluid (CSF), and saliva by real-time quantitative PCR in two patients, one with a history of nasopharyngeal carcinoma, and the other having a case of viral encephalitis. EBV was detected only in saliva, whole blood in both patients, and CSF in the second case tested negative. This case series illustrates the importance of testing for EBV DNAemia in saliva by real-time polymerase chain reaction (PCR) to rule in a diagnosis of EBV.

Development of a Rapid Epstein-Barr Virus Detection System Based on Recombinase Polymerase Amplification and a Lateral Flow Assay.

The quality of cellular products used in biological research can directly impact the ability to obtain accurate results. Epstein-Barr virus (EBV) is a latent virus that spreads extensively worldwide, and cell lines used in experiments may carry EBV and pose an infection risk. The presence of EBV in a single cell line can contaminate other cell lines used in the same laboratory, affecting experimental results. We developed three EBV detection systems: (1) a polymerase chain reaction (PCR)-based detection system, (2) a recombinase polymerase amplification (RPA)-based detection system, and (3) a combined RPA-lateral flow assay (LFA) detection system. The minimum EBV detection limits were 1 × 103 copy numbers for the RPA-based and RPA-LFA systems and 1 × 104 copy numbers for the PCR-based system. Both the PCR and RPA detection systems were applied to 192 cell lines, and the results were consistent with those obtained by the EBV assay methods specified in the pharmaceutical industry standards of the People's Republic of China. A total of 10 EBV-positive cell lines were identified. The combined RPA-LFA system is simple to operate, allowing for rapid result visualization. This system can be implemented in laboratories and cell banks as part of a daily quality control strategy to ensure cell quality and experimental safety and may represent a potential new technique for the rapid detection of EBV in clinical samples.

Multiple cross displacement amplification combined with nanoparticle-based lateral flow biosensor for rapid and sensitive detection of Epstein-Barr virus.

Epstein-Barr virus (EBV) is a highly dangerous virus that is globally prevalent and closely linked to the development of nasopharyngeal cancer (NPC). Plasma EBV DNA analysis is an effective strategy for early detection, prognostication and monitoring of treatment response of NPC. Here, we present a novel molecular diagnostic technique termed EBV-MCDA-LFB, which integrates multiple cross displacement amplification (MCDA) with nanoparticle-based lateral flow (LFB) to enable simple, rapid and specific detection of EBV. In the EBV-MCDA-LFB system, a set of 10 primers was designed for rapidly amplifying the highly conserved tandem repeat BamHI-W region of the EBV genome. Subsequently, the LFB facilitate direct assay reading, eliminating the use of extra instruments and reagents. The outcomes showed that the 65°C within 40 minutes was the optimal reaction setting for the EBV-MCDA system. The sensitivity of EBV-MCDA-LFB assay reached 7 copies per reaction when using EBV recombinant plasmid, and it showed 100% specificity without any cross-reactivity with other pathogens. The feasibility of the EBV-MCDA-LFB method for EBV detection was successfully validated by 49 clinical plasma samples. The complete detection process, consisting of rapid template extraction (15 minutes), MCDA reaction (65°C for 40 minutes), and LFB result reading (2 minutes), can be finalized within a 60-minutes duration. EBV-MCDA-LFB assay designed here is a fast, extremely sensitive and specific technique for detecting EBV in field and at the point-of-care (PoC), which is especially beneficial for countries and regions with a high prevalence of the disease and limited economic resources.

Microfluidic bead-based biosensor: Ultrasensitive ctDNA detection based on duplex-functional split-DNAzyme and dendritic enzyme-free signal amplification

Circulating tumor DNA (ctDNA) is a crucial cancer biomarker for early or noninvasive monitoring, which is essential for developing ultrasensitive and selective assays in cancer diagnosis and treatment. Herein, a cascade signal amplification of duplex-functional split-DNAzyme and dendritic probes was proposed for ultrasensitive and specific detection of nasopharyngeal carcinoma-associated Epstein-Barr virus (EBV) DNA on microfluidic microbead array chips. With the assistance of Pb2+, the duplex-functional split-DNAzyme recognizes EBV DNA and then rapidly cleaves the substrate strand. Subsequently, the released target could be recycled, and its exposed capture probe, triggered the dendritic enzyme-free signal amplification. As the enhanced mass transfer capability, target recycling, and dendritic DNA structure signal amplification inherent to microfluidic bead arrays were integrated, it achieved an excellent detection limit of 0.36 fM and a wide linear range of 1 fM∼103 fM. Further, it was applied to content detect simulated samples of EBV DNA, recovery ranged from 97.2 % to 108.1 %, and relative standard deviation (RSD) from 3.3 % to 5.9 %, exhibiting satisfactory recovery results. The developed microfluidic biosensor was a high-sensitivity and anti-interference system for ctDNA analysis, with minimal reagent volumes (microlitres) required. Thus, it is a promising platform for ctDNA at the lowest level at their earliest incidence.

Patterns and Prognosis of Local Recurrence of Nasopharyngeal Carcinoma after Intensity-modulated Radiotherapy.

Objective: To investigate the patterns of local failure and prognosis in patients with locally recurrent nasopharyngeal carcinoma (rNPC) after primary intensity-modulated radiotherapy (IMRT). Methods: The data of 298 patients with locally rNPC after IMRT were retrospectively analyzed. Magnetic resonance images of the initial and recurrent tumors were reviewed and, for patients with extra-nasopharyngeal local recurrence, the gross tumor volume of local recurrence was transferred to the original IMRT plan for dosimetry analysis. Significant prognostic factors for overall survival (OS) were selected by multivariate Cox regression analysis. Results: The commonest recurrence sites were the nasopharynx (93%, 277/298) and skull base (53.7%, 160/298). Of the 21 patients with extra-nasopharyngeal recurrence (19 cases valid), 12 had in-field failures, 4 had marginal failures, and 3 had out-field failures. The ethmoid sinus (57.1%, 4/7) and nasal cavity (28.6%, 2/7) were the most frequent sites of marginal and out-field failures. After median follow-up of 37 months, the 3-year and estimated 5-year OS rates were 57.3% and 41.7%, respectively. Multivariate analysis showed that age, recurrence interval, plasma Epstein-Barr virus (EBV) DNA level, and recurrent T stage were independent prognostic factors for OS. Conclusions: Local failure after IMRT occurs most commonly in the nasopharynx and skull base. In patients with extra-nasopharyngeal recurrence, in-field failure remains the main failure pattern, and marginal and out-field failures mainly occur in the ethmoid sinus and nasal cavity. Elder age, shorter recurrence interval, detectable plasma EBV DNA, and advanced recurrent T stage are negative predictors of OS in patients with rNPC.

Development of a Rapid Epstein–Barr Virus Detection System Based on Recombinase Polymerase Amplification and a Lateral Flow Assay

Development of a Rapid Epstein–Barr Virus Detection System Based on Recombinase Polymerase Amplification and a Lateral Flow Assay