Detection of free DNA based on metal-enhanced fluorescence triggered by CRISPR-Cas12a and colorimetric analysis.

The CRISPR-Cas system has been found to be extremely sensitive and there is an urgent demand to extend its potential in bioassays. Herein, we developed a novel nanobiosensor to detect the human papillomavirus 16 genes (HPV-16 DNA), which is triggered by CRISPR-Cas12a to amplify the fluorescence signal by metal-enhanced fluorescence (CAMEF). Along with the changing of the fluorescence signal, the aggregation of the substrate of MEF also leads to a change in the color of the mixture solution, enabling dual signal detection with the fluorescence and the naked eye. Furthermore, the designed CAMEF probe was verified to detect the HPV-16 DNA accurately and reliably in biological samples. Triggered by the CRISPR system, the designed CAMEF probe allows quantitative detection of the HPV-16 DNA in the wide range of 10-500 pM. Owing to the MEF, the fluorescence signal of the CAMEF probe was significantly amplified with the detection limit as low as 1 pM. Besides, we can determine the concentration of HPV-16 DNA simply by the naked eye, which also drastically reduces the possibility of false-positive signals. Theoretically, the target ssDNA could be any strand of DNA obtained by designing the crRNA sequence in the CRISPR-Cas system. We believe that the designed CAMEF sensor can present a reliable approach for the accurate detection of low amounts of target ssDNA in complex biological samples.

Impact of a carrageenan gel on viral load of genital human papillomavirus infections in sexually active women: Findings from the Carrageenan-gel Against Transmission of Cervical Human papillomavirus (CATCH) trial.

Previous research has shown that women's use of a carrageenan gel reduces the risk of acquiring genital human papillomavirus (HPV) infections but does not help to clear existing ones. Although gel use may not result in complete clearance, it may decrease the viral load of HPV infections. We tested this hypothesis in the Carrageenan-gel Against Transmission of Cervical Human papillomavirus (CATCH) randomized controlled trial. Participants of the CATCH study were selected for viral load testing if they had completed the first four study visits and tested positive for HPV42 or HPV51 in at least one of these visits. HPV42 and HPV51 were chosen as they were among the most abundant low- and high-risk types, respectively, in the study sample. We measured viral load with a type-specific real-time polymerase chain reaction. Results were displayed using summary statistics. Of 461 enrolled participants, 39 were included in the HPV42 analysis set and 56 in the HPV51 analysis set. The median time between visits 1 and 4 was 3.7 months. The viral load (copies/cell) of HPV42 ranged from <0.001 to 13 434.1, and that of HPV51 from <0.001 to 967.1. The net median change in HPV42 viral load over all four visits was -1.04 copies/cell in the carrageenan and -147 copies/cell in the placebo arm (Wilcoxon rank sum test, p = 0.26). There was no net median change in HPV51 viral load over all four visits in either arm (p = 0.45). The use of a carrageenan-based gel is unlikely to reduce the viral load of HPVs 42 or 51.

Establishing a screening strategy for non-discriminatory reactive blood donors by constructing a predictive model of hepatitis B virus infection status from a single blood center in China.

Background: When employing the transcription-mediated amplification method for screening blood donors, there are some non-discriminatory reactive results which are screening assay reactive but HBV-DNA discriminatory assay negative. This raises concerns regarding the possibility of false positives among donors, which may lead to permanent deferral of blood donors and affect blood supply. This study aimed to elucidate the infection status of these non-discriminatory reactive blood donors and develop and validate a model to predict individualized hepatitis B status to establish an optimal screening strategy. Methods: Supplementary tests were conducted on initial non-discriminating reactive donations to determine their HBV infection status, including repeat testing, viral load, serological marker detection, and follow-up. Primary clinical variables of the donors were recorded. Based on the Akaike information criterion, a stepwise forward algorithm was used to identify the predictive factors for information and construct a predictive model. The optimal screening strategy was determined through cost-effectiveness analysis. Results: At the Blood Center of Zhejiang Province, 435 cases of initial non-discriminatory reactive donations were collected over two successive periods and sub-categorized through repeated testing into the following three groups: non-repeated positive group, non-discriminated positive group, and non-repeated HBV-DNA positive group. The HBV discriminatory rate increased after repeated testing (110/435, 25.29%). According to supplementary tests, the HBV-DNA positivity rate was 65.52% (285/435), and occult HBV infection was a significantly different among groups (χ2 = 93.22, p < 0.01). The HBV serological markers and viral load in the non-repeated positive group differed from those in the other two groups, with a lower viral load and a higher proportion of false positives. The predictive model constructed using a stepwise forward algorithm exhibited high discrimination, good fit, high calibration, and effectiveness. A cost-effectiveness analysis indicated that utilizing repeated discriminatory testing and the predictive model is an extremely beneficial screening approach for non-discriminatory reactive blood donors. Conclusion: Nearly two-third (65.52%) of the non-discriminatory reactive blood donors were HBV-DNA positive. Our innovative approach of constructing a predictive model as a supplementary screening strategy, combined with repeated discriminatory experiments, can effectively identify the infection status of non-discriminatory reactive blood donors, thereby increasing the safety of blood transfusions.

Non-Detection of HCMV Total Genomic DNA in Human Glioma Cells Genome.

AIM: To demonstrate if the human cytomegalovirus (HCMV) genome, that is involved in the pathogenesis of gliomas, is part of the genomic DNA of glioma cells or not. MATERIAL AND METHODS: The study included U87MG glioblastoma cell culture and tumor samples from glioma patients. The genomic DNA of tumor samples and U87MG cells were extracted and real-time quantitative PCR was used to assess the presence of the human cytomegalovirus genomic DNA. RESULTS: Consequently, HCMV positivity was not detected in the tumor and cell line genomic DNA under the aforementioned experimental conditions. CONCLUSION: We found that the genomic DNA of all the samples was negative for HCMV genomic DNA. Thus, HCMV could not be detected in human glioma tumors and we put forward that HCMV genomic DNA was not incorporated into the genomic DNA of glioma cells. Thus, total viral DNA is not involved in the pathogenesis of glioma; however, small viral particles or specific genes might be incorporated into the genomic DNA of glioma cells, leading to cancer development. This prompts further studies for verification.

The first detection of mpox virus DNA from wastewater in China.

Wastewater monitoring may be a valuable early surveillance tool for studying mpox virus (MPXV) circulation in China, a country with high population density and very few mpox patients. To evaluate the effectiveness of wastewater monitoring for MPXV in detecting local hidden transmission of the epidemic in the early period, the Chinese Center for Disease Control and Prevention initiated a wastewater monitoring program for MPXV in China in July 2023. To enhance the monitoring sensitivity of the program, an MPXV monitoring point was established in a gathering place of high-risk mpox population. Three different concentration methods, PEG precipitation, ultrafiltration, and magnetic beads method were evaluated and compared. Due to its high recovery efficiency, low limit of detection, and high degree of automation, the magnetic beads method was selected for the daily surveillance of MPXV in wastewater. On September 5, 2023, MPXV DNA was detected at the MPXV monitoring point in Zibo City, marking the first instance of MPXV detection of MPXV in wastewater in China. Next-generation sequencing was conducted on the MPXV genome obtained from the positive wastewater, positive environmental samples, and the single case of mpox in Zibo in September. The results showed that the genotypes of these three genomes were different but all belong to the IIb branch of the C.1 lineage, indicating a probably hidden transmission of mpox. Wastewater monitoring is potentially an effective early surveillance tool for tracking the spread of MPXV in areas with high population density and very few mpox patients.

Preoperative Circulating Tumor HPV DNA and Oropharyngeal Squamous Cell Disease.

Importance: The utility of preoperative circulating tumor tissue-modified viral human papillomavirus DNA (TTMV-HPV DNA) levels in predicting human papillomavirus (HPV)-associated oropharyngeal squamous cell carcinoma (HPV+ OPSCC) disease burden is unknown. Objective: To determine if preoperative circulating tumor HPV DNA (ctHPVDNA) is associated with disease burden in patients with HPV+ OPSCC who have undergone transoral robotic surgery (TORS). Design, Setting, and Participants: This cross-sectional study comprised patients with HPV+ OPSCC who underwent primary TORS between September 2021 and April 2023 at one tertiary academic institution. Patients with treatment-naive HPV+ OPSCC (p16-positive) and preoperative ctHPVDNA levels were included, and those who underwent neck mass excision before ctHPVDNA collection were excluded. Main Outcomes and Measures: The main outcome was the association of increasing preoperative ctHPVDNA levels with tumor size and lymph node involvement in surgical pathology. The secondary outcome was the association between preoperative ctHPVDNA levels and adverse pathology, which included lymphovascular invasion, perineural invasion, or extranodal extension. Results: A total of 70 patients were included in the study (65 men [93%]; mean [SD] age, 61 [8] years). Baseline ctHPVDNA levels ranged from 0 fragments/milliliter of plasma (frag/mL) to 49 452 frag/mL (median [IQR], 272 [30-811] frag/mL). Overall, 58 patients (83%) had positive results for ctHPVDNA, 1 (1.4%) had indeterminate results, and 11 (15.6%) had negative results. The sensitivity of detectable ctHPVDNA for identifying patients with pathology-confirmed HPV+ OPSCC was 84%. Twenty-seven patients (39%) had pathologic tumor (pT) staging of pT0 or pT1, 34 (49%) had pT2 staging, and 9 patients (13%) had pT3 or pT4 staging. No clinically meaningful difference between detectable and undetectable preoperative ctHPVDNA cohorts was found for tumor size or adverse pathology. Although the median preoperative ctHPVDNA appeared to be higher in pT2 through pT4 stages and pN1 or pN2 stages, effect sizes were small (pT stage: η2, 0.002 [95% CI, -1.188 to 0.827]; pN stage: η2, 0.043 [95% CI, -0.188 to 2.600]). Median preoperative log(TTMV-HPV DNA) was higher in active smokers (8.79 [95% CI, 3.55-5.76]), compared with never smokers (5.92 [95% CI, -0.97 to 1.81]) and former smokers (4.99 [95% CI, 0.92-6.23]). Regression analysis did not show an association between tumor dimension or metastatic lymph node deposit size and preoperative log(TTMV-HPV DNA). After univariate analysis, no association was found between higher log(TTMV-HPV DNA) levels and adverse pathology. Conclusions and Relevance: In this cross-sectional study, preoperative ctHPVDNA levels were not associated with disease burden in patients with HPV+ OPSCC who underwent TORS.

[Effect of HBV DNA load on the safety and prognosis of systematic therapy in advanced hepatocellular carcinoma].

Objective: To study the effect of hepatitis B virus (HBV) infection on the occurrence of liver damage, HBV reactivation (HBVr) and the influence of HBVr on the prognosis of patients with advanced hepatocellular carcinoma (HCC) receiving systemic therapy. Methods: The clinical data of 403 patients with HBV-related HCC at the Department of Infectious Diseases, The Third Affiliated Hospital of Sun Yat-Sen University et al, from July 2018 to December 2020 were collected. The incidence of liver damage and HBVr during systematic therapy, and the influence of HBVr on survival prognosis were analyzed. Results: Of the 403 patients, 89.1% were male (n=359), with a median age of 51 years (51.5±12.1). Before propensity score matching (PSM), the proportion of patients with cirrhosis, TNM and advanced BCLC stage was higher in high HBV-DNA (baseline HBV-DNA>1000 U/ml, n=147) group comparing with the low HBV-DNA (baseline HBV DNA≤1000 u/ml, n=256) group (P<0.05). There was no significant difference in baseline indexes between the two groups after PSM. In 290 patients after PSM, there was no significant difference in the incidence of liver damage and HBVr between high HBV-DNA group and low HBV-DNA group (P>0.05). Survival analysis was performed on 169 patients with survival data, the median overall survival (OS) was found to be 11.49 months (95%CI: 7.77-12.89) and 16.65 months (95%CI: 10.54-21.99, P=0.008) in the high and low HBV-DNA groups, respectively. And median progression-free survival (PFS) was 7.41 months (95%CI: 5.06-8.67) and 10.55 months (95%CI: 6.72-13.54, P=0.038), respectively, with a statistically significant difference. There were no differences in overall survival (OS) and progression-free survival (PFS) between patients with and without HBVr and those with or without liver damage (P>0.05). Conclusions: HBV-DNA levels above 1 000 U/ml before systemic therapy do not increase the risk of liver damage or HBVr during systemic therapy in patients with HBV-related hepatocellular carcinoma, and such patients can safely receive systemic therapy.

Improving polymerase chain reaction diagnostic rates for herpes simplex keratitis: results of a pilot study.

Background: Laboratory confirmation is crucial for diagnosis and management of herpes simplex virus (HSV) keratitis. However, the sensitivity of polymerase chain reaction (PCR) in keratitis is low (25%) compared with that of mucocutaneous disease (75%). We developed an educational intervention aimed at improving the diagnostic yield of PCR. Methods: The medical records of keratitis cases seen at the emergency department of a London tertiary ophthalmic referral hospital over two distinct periods, before and after an educational program on swab technique, were reviewed retrospectively. Results: A total of 252 HSV cases were included. Increases in the laboratory-confirmed diagnosis of HSV-1 were observed, in both first presentations (11.1%-57.7%) and recurrent cases (20%-57.6%). The rate of positive HSV-1 PCR in eyes with an epithelial defect increased from 19% pre-intervention to 62% post intervention. Notably, 3% were positive for varicella zoster virus DNA, and there was a single case of Acanthamoeba keratitis. Conclusion: Our results suggest that, with proper swabbing technique, PCR may be more sensitive than previously reported.

Human Papillomavirus Is Rare and Does Not Correlate with p16INK4A Expression in Non-Small-Cell Lung Cancer in a Jordanian Subpopulation.

Background and Objectives: Human papillomavirus (HPV) was previously investigated in lung cancer with wide inter-geographic discrepancies. p16INK4a has been used as a surrogate for detecting high-risk HPV (HR-HPV) in some cancer types. This study assessed the evidence of HPV in non-small-cell lung cancer (NSCLC) among Jordanian patients, investigated the expression of p16INK4a, and evaluated its prognostic value and association with HPV status. Materials and Methods: The archived samples of 100 patients were used. HPV DNA detection was performed by real-time polymerase chain reaction (RT-PCR). p16INK4a expression was assessed by immunohistochemistry (IHC). The Eighth American Joint Committee on Cancer protocol (AJCC) of head and neck cancer criteria were applied to evaluate p16INK4a positivity considering a moderate/strong nuclear/cytoplasmic expression intensity with a distribution in ≥75% of cells as positive. Results: HPV DNA was detected in 5% of NSCLC cases. Three positive cases showed HR-HPV subtypes (16, 18, 52), and two cases showed the probable HR-HPV 26 subtype. p16INK4a expression was positive in 20 (20%) NSCLC cases. None of the HPV-positive tumors were positive for p16INK4a expression. A statistically significant association was identified between p16INK4a expression and the pathological stage (p = 0.029) but not with other variables. No survival impact of p16INK4a expression was detected in NSCLC cases as a group; however, it showed a statistically significant association with overall survival (OS) in squamous cell carcinoma (SqCC) cases (p = 0.033). Conclusions: This is the first study to assess HPV and p16INK4a expression in a Jordanian population. HPV positivity is rare in NSCLC among a Jordanian subpopulation. P16 INK4a reliability as a surrogate marker for HPV infection in lung cancer must be revisited.

Low-Triggering-Potential and Narrow-Potential-Window Electrochemiluminescence of Silver Nanoclusters for Gene Assay.

A low-triggering potential and a narrow-potential window are anticipated to decrease the electrochemical interference and cross talk of electrochemiluminescence (ECL). Herein, by exploiting the low oxidative potential (0.82 V vs Ag/AgCl) of dihydrolipoic acid-capped sliver nanoclusters (DHLA-AgNCs), a coreactant ECL system of DHLA-AgNCs/hydrazine (N2H4) is proposed to achieve efficient and oxidative-reduction ECL with a low-triggering potential of 0.82 V (vs Ag/AgCl) and a narrow-potential window of 0.22 V. The low-triggering-potential and narrow-potential-window nature of ECL can be primarily preserved upon labeling DHLA-AgNCs to probe DNA and immobilizing DHLA-AgNCs onto the Au surface via sandwiched hybridization, which eventually enables a selective ECL strategy for the gene assay at +0.82 V. This gene assay strategy can sensitively determine the gene of human papillomavirus from 10 to 1000 pM with a low limit of detection of 5 pM (S/N = 3) and would open a way to improve the applied ECL bioassay.

Enhanced CRISPR/Cas12a-based quantitative detection of nucleic acids using double emulsion droplets.

Pairing droplet microfluidics and CRISPR/Cas12a techniques creates a powerful solution for the detection and quantification of nucleic acids at the single-molecule level, due to its specificity, sensitivity, and simplicity. However, traditional water-in-oil (W/O) single emulsion (SE) droplets often present stability issues, affecting the accuracy and reproducibility of assay results. As an alternative, water-in-oil-in-water (W/O/W) double emulsion (DE) droplets offer superior stability and uniformity for droplet digital assays. Moreover, unlike SE droplets, DE droplets are compatible with commercially available flow cytometry instruments for high-throughput analysis. Despite these advantages, no study has demonstrated the use of DE droplets for CRISPR-based nucleic acid detection. In our study, we conducted a comparative analysis to assess the performance of SE and DE droplets in quantitative detection of human papillomavirus type 18 (HPV18) DNA based on CRISPR/Cas12a. We evaluated the stability of SEs and DEs by examining size variation, merging extent, and content interaction before and after incubation at different temperatures and time points. By integrating DE droplets with flow cytometry, we achieved high-throughput and high-accuracy CRISPR/Cas12a-based quantification of target HPV18 DNA. The DE platform, when paired with CRISPR/Cas12a and flow cytometry techniques, emerges as a reliable tool for absolute quantification of nucleic acid biomarkers.

Silica Nanowires-Filled Glass Microporous Sensor for the Ultrasensitive Detection of Deoxyribonucleic Acid.

DNA carries genetic information and can serve as an important biomarker for the early diagnosis and assessment of the disease prognosis. Here, we propose a bottom-up assembly method for a silica nanowire-filled glass microporous (SiNWs@GMP) sensor and develop a universal sensing platform for the ultrasensitive and specific detection of DNA. The three-dimensional network structure formed by SiNWs provides them with highly abundant and accessible binding sites, allowing for the immobilization of a large amount of capture probe DNA, thereby enabling more target DNA to hybridize with the capture probe DNA to improve detection performance. Therefore, the SiNWs@GMP sensor achieves ultrasensitive detection of target DNA. In the detection range of 1 aM to 100 fM, there is a good linear relationship between the decrease rate of current signal and the concentration of target DNA, and the detection limit is as low as 1 aM. The developed SiNWs@GMP sensor can distinguish target DNA sequences that are 1-, 3-, and 5-mismatched, and specifically recognize target DNA from complex mixed solution. Furthermore, based on this excellent selectivity and specificity, we validate the universality of this sensing strategy by detecting DNA (H1N1 and H5N1) sequences associated with the avian influenza virus. By replacing the types of nucleic acid aptamers, it is expected to achieve a wide range and low detection limit sensitive detection of various biological molecules. The results indicate that the developed universal sensing platform has ultrahigh sensitivity, excellent selectivity, stability, and acceptable reproducibility, demonstrating its potential application in DNA bioanalysis.

Unusual cerebral intraventricular hemorrhage and cardiomyopathy related to congenital cytomegalovirus from non-primary maternal infection: a case report.

BACKGROUND: Congenital cytomegalovirus (cCMV) infection, resulting from non-primary maternal infection or reactivation during pregnancy, can cause serious fetal abnormalities, complications in the immediate neonatal period, and severe sequelae later in childhood. Maternal non-primary cytomegalovirus infection in pregnancy is transmitted to the fetus in 0.5-2% of cases (1). CASE PRESENTATION: An African full term male newbornwas delivered by emergency caesarean section. Due to signs of asphyxia at birth and clinical moderate encephalopathy, he underwent therapeutic hypothermia. Continuous full video-electroencephalography monitoring showed no seizures during the first 72 h, however, soon after rewarming, he presented refractory status epilepticus due to an intracranial hemorrhage, related to severe thrombocytopenia. The patient also presented signs of sepsis (hypotension and signs of reduced perfusions). An echocardiography revealed severe cardiac failure with an ejection fraction of 33% and signs suggestive of cardiomyopathy. Research for CMV DNA Polymerase Chain Reaction (PCR) on urine, blood, cerebrospinal fluid, and nasopharyngeal secretions was positive.The mother had positive CMV IgG with negative IgM shortly before pregnancy. Serology for CMV was therefore not repeated during pregnancy, but CMV DNA performed on the Guthrie bloodspot taken at birth yielded a positive result, confirming the intrauterine transmission and congenital origin of the infection. The baby was discharged in good general condition and follow up showed a normal neurodevelopmental outcome at 9 months. CONCLUSION: Although uncommon, congenital cytomegalovirus infection should be included in the differential diagnosis of intraventricular hemorrhage and cardiomyopathy. Furthermore, this case highlights the possible severity of congenital cytomegalovirus infection, even in cases of previous maternal immunity.

A quantitative assay for the assessment of cutaneous human papillomaviruses and polyomaviruses over time: A proof-of-concept in two patients with atopic dermatitis and psoriasis.

The human skin virome, unlike commensal bacteria, is an under investigated component of the human skin microbiome. We developed a sensitive, quantitative assay to detect cutaneous human resident papillomaviruses (HPV) and polyomaviruses (HPyV) and we first used it to describe these viral populations at the skin surface of two patients with atopic dermatitis (AD) and psoriasis (PSO). We performed skin swabs on lesional and non-lesional skin in one AD and one PSO patient at M0, M1 and M3. After extraction, DNA was amplified using an original multiplex PCR technique before high throughput sequencing (HTS) of the amplicons (named AmpliSeq-HTS). Quantitative results were ultimately compared with monoplex quantitative PCRs (qPCRs) for previously detected viruses and were significantly correlated (R2 = 0.95, ρ = 0.75). Fifteen and 13 HPV types (mainly gamma and beta-HPVs) or HPyV species (mainly Merkel Cell Polyomavirus (MCPyV)) were detected on the skin of the AD and PSO patients, respectively. In both patients, the composition of the viral flora was variable across body sites but remained stable over time in non-lesional skin samples, mostly colonized with gamma-papillomaviruses. In lesional skin samples, beta-papillomaviruses and MCPyV were the major components of a viral flora more prone to vary over time especially with treatment and subsequent clinical improvement. We believe this method might be further used in extensive studies to further enhance the concept of an individual cutaneous viral fingerprint and the putative role of its alterations through various skin diseases and their treatments.

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

INTRODUCTION: The objective was to investigate the correlation between Epstein-Barr virus (EBV) and human papillomavirus (HPV) infection in the development of benign thyroid lesions. MATERIAL AND METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Validation of the VisionArray® Chip Assay for HPV DNA Testing in Histology Specimens of Oropharyngeal Squamous Cell Carcinoma.

BACKGROUND: The detection of human papillomavirus (HPV) has several implications in the diagnostic work-up and management of oropharyngeal squamous cell carcinoma (OPSCC). The choice of HPV detection assay and testing algorithms differ across institutions and vary in cost, detection targets, technical feasibility, and turnaround time. In this study, we aimed to validate the VisionArray® HPV Chip for formalin-fixed and paraffin-embedded (FFPE) samples of OPSCC using the previously applied standard pan-HPV DNA PCR assay as a reference. METHODS: The validation cohort consisted of FFPE tissue samples from patients previously diagnosed with HPV DNA-positive OPSCC (n = 80), HPV DNA-negative OPSCC (n = 21), and a benign group of tumor samples consisting of Warthin's tumors (n = 20) and branchial cleft cysts of the lateral neck (n = 14). All samples were tested with p16 immunohistochemistry, pan-HPV DNA PCR, and the VisionArray® HPV Chip. RESULTS: The overall sensitivity and specificity of the VisionArray® HPV Chip assay were 100% [95% CI 95.5%; 100.0%] and 96.3% [95% CI 87.3%; 99.6%] and the positive predictive value and negative predictive value were 97.6% [95% CI 91.5%; 99.7%] and 100% [95% CI 93.2%; 100%], respectively. CONCLUSIONS: The VisionArray® HPV Chip assay can be recommended for high-risk HPV testing in FFPE tissue samples from OPSCC, providing both a fast and simultaneous genotyping for 41 clinically relevant HPV types.

Evaluation of Congenital Cytomegalovirus Infection in Pregnant Women Admitted to a University Hospital in Istanbul.

Cytomegalovirus (CMV) can cause serious complications in immunocompromised individuals and fetuses with congenital infections. These can include neurodevelopmental impairments and congenital abnormalities in newborns. This paper emphasizes the importance of concurrently evaluating ultrasonography findings and laboratory parameters in diagnosing congenital CMV infection. To examine the prenatal characteristics of CMV DNA-positive patients, we assessed serum and amniotic fluid from 141 pregnant women aged 19-45 years, each with fetal anomalies. ELISA and PCR tests, conducted in response to these amniocentesis findings, were performed at an average gestational age of 25 weeks. Serological tests revealed that all 141 women were CMV IgG-positive, and 2 (1.41%) had low-avidity CMV IgG, suggesting a recent infection. CMV DNA was detected in 17 (12.05%) amniotic fluid samples using quantitative PCR. Of these, 82% exhibited central nervous system abnormalities. Given that most infections in pregnant women are undetectable and indicators non-specific, diagnosing primary CMV in pregnant women using clinical findings alone is challenging. We contend that serological tests should not be the sole means of diagnosing congenital CMV infection during pregnancy.

Prevalence of BK and JC Polyomaviruses among Patients with Breast Cancer.

INTRODUCTION: Breast cancer, a pervasive invasive carcinoma among women globally, afflicts approximately 12% of women worldwide. Previous studies have indicated that certain viruses, including oncogenic viruses such as polyomaviruses BK and JC, may play a role in the development of breast cancer. In light of this, the present study endeavors to assess the incidence of BKV and JCV virus in breast cancer patients. MATERIALS AND METHODS: One hundred formalin-fixed paraffin-embedded tissue samples were procured and subjected to deparaffinize by xylene, followed by DNA extraction through the phenol-chloroform methodology. Detection and genotyping of BKV and JCV were carried out utilizing specific primers via PCR analysis. RESULTS: Merely 2 out of 100 (2%) ductal carcinoma in situ with grade 2 specimens exhibited positivity for BK virus genotype IV, whereas JC virus DNA was not discerned across all the samples. DISCUSSION: The findings of the current investigation demonstrate that there was an absence of JC virus detection in the breast biopsy. Additionally, a small fraction of patients diagnosed with ductal carcinoma exhibited a low prevalence of genotype IV polyomavirus BK at a rate of 2%. However, in order to gain a more comprehensive understanding of the incidence of BKV and JCV in breast cancer, a substantial number of breast samples must undergo investigation.

Human papillomavirus (HPV) load is higher in HPVDNA/p16 positive than in HPVDNA positive/p16 negative oropharyngeal squamous cell carcinoma but does not differ significantly between various subsites or correlate to survival.

OBJECTIVE: Patients with human papillomavirus DNA positive (HPVDNA+) and p16ink4a overexpressing (p16+) oropharyngeal squamous cell carcinoma (OPSCC), especially those with cancer in the tonsillar and base of tongue subsites as compared to other OPSCC subsites have a better outcome than those with only HPVDNA+ or only p16+ cancer. Likewise having a high viral load has been suggested to be a positive prognostic factor. We therefore hypothesized, that HPV viral load could vary depending on OPSCC subsite, as well as with regard to whether the cancer was HPVDNA+ and p16+, or only HPVDNA+, or only p16+ and that this affected outcome. MATERIAL AND METHODS: To address these issues HPV viral load was determined by HPV digital droplet (dd) PCR in tumor biopsies with previously known HPVDNA/p16 status from 270 OPSCC patients diagnosed 2000-2016 in Stockholm, Sweden. More specifically, of these patients 235 had HPVDNA+/p16+, 10 had HPVDNA+/p16-, 13 had HPVDNA-/p16+ and 12 had HPVDNA-/p16- cancer. RESULTS: We found that HPVDNA+/p16+ OPSCC had a significantly higher viral load than HPVDNA+/p16- OPSCC. Moreover, there was a tendency for a higher viral load in the tonsillar and base of tongue OPSCC subsites compared to the other subsites and for a low viral load to correlate to a better clinical outcome but none of these tendencies reached statistical significance. CONCLUSION: To conclude, the mean viral load in HPVDNA+/p16+ OPSCC was higher than in HPVDNA+/p16- OPSCC, but there was no statistically significant difference in viral load depending on OPSCC subsite or on clinical outcome.

Rapid immunoassay for dual-mode detection of HPV16 and HPV18 DNA based on Au@PdPt nanoparticles.

Cervical cancer (CC) remains one of the most severe global health challenges affecting women, primarily due to persistent infection with high-risk human papillomavirus (HPV) subtypes, particularly with HPV16 and HPV 18. Effective detection of these high-risk HPV strains is crucial for CC prevention. Current screening programs for HPV DNA include PCR and in situ hybridization, which are accurate and sensitive. However, these approaches demand a high level of expertise, along with expensive instruments and consumables, thus hindering their widespread use. Therefore, there is a compelling demand to develop an efficient, straightforward, and cost-effective method. Herein, we propose a lateral flow immunoassay (LFIA) method based on Au@PdPt nanoparticles for the simultaneous detection and genotyping of HPV16 and HPV18 within 15 min. This innovative approach allows for qualitative assessment by the naked eye and enables semi-quantitative detection through a smartphone. In this study, under optimal conditions, the qualitative visual limits of detection (vLOD) for HPV16 and HPV18 reached 0.007 nM and 0.01 nM, respectively, which were 32-fold and 20-fold more sensitive than conventional AuNPs-LFIA for HPV16 and HPV18, respectively. Meanwhile, semi-quantitative limits of detection (qLOD) for HPV16 and HPV18 were 0.05 nM and 0.02 nM, respectively. In conclusion, our formulated approach represents a significant step forward in HPV detection and genotyping, with the potential to enhance accessibility and effectiveness in the early diagnosis of CC at the point of care and beyond.