Search Results (1,032)

Wastewater surveillance emerged as a critical public health tool during the COVID-19 pandemic, enabling early detection of community-level pathogen circulation independent of clinical testing. Its ability to capture signals from both symptomatic and asymptomatic individuals highlighted the importance of optimizing sampling methodologies to improve sensitivity and reliability. A key question is whether the several-fold increase in SARS-CoV-2 detectability observed when using passive tampon swab sampling compared with paired grab samples also applies to other respiratory viruses, including influenza A (including its avian influenza H5N1 subtype), influenza B, and respiratory syncytial virus (RSV). We collected 24 h passive swab samples with same-day grab samples from Detroit sewersheds, concentrated and purified nucleic acids, and using RT-ddPCR, quantified respiratory syncytial virus, SARS-CoV-2, influenza A, influenza B, and H5N1 influenza A viruses using markers RSV, SC2, InfA, InfB, and H5, respectively. Samples testing positive for H5 (marker for H5N1 influenza A) were further analyzed by targeted PCR and amplicon sequencing. Across three sites, median 24 h swab:grab ratios of virus copies were 7.0 for RSV, 9.2 for SC2, 9.9 for InfA, and 3.6 for InfB. A 239 bp hemagglutinin sequence from a sample with a strong H5 signal (795 copies/10 mL) had 100% identity to avian influenza viruses from Canada geese. Twenty-four-hour swab sampling greatly improves viral detectability across diverse targets and enabled the first confirmed detection of H5 in Detroit wastewater. Combined with magnetic bead purification, the overall sensitivity gain over conventional PEG-NaCl-Qiagen methods is approximately 36-fold, enabling earlier warning of community pathogens than grab samples. By integrating 24 hour passive swab sampling with high-efficiency nucleic acid purification, we expand the sensitivity of wastewater surveillance to enable detection and confirmation of low-abundance pathogens like avian influenza (H5). Full article

Determining the characteristics of hepatitis B virus (HBV) infection in the healthy population and evaluating the effectiveness of detection strategies will facilitate the optimization of hepatitis B screening strategies in the community and accelerate the elimination of HBV infection in China by the end of 2030. Hepatitis B surface antigen (HBsAg)-electrochemiluminescence immunoassays (ECLIAs), HBsAg-rapid diagnostic tests (RDTs), and HBV DNA-nucleic acid tests (NATs) were performed on serum samples from 2721 community-based healthy participants in Guangdong Province. The screening performance of the RDT and NAT and the distribution characteristics of HBsAg and HBV DNA were evaluated. The prevalence rates of HBsAg-ECLIA, HBsAg-RDT and HBV DNA-NAT in Guangdong Province were 6.10% (95% CI: 5.26~7.06), 4.96% (95% CI: 4.21~5.84) and 6.55% (95% CI: 5.64~7.49), respectively, and the prevalence rates for the three methods for individuals aged over 30 years were 11.18%, 10.92% and 12.57%, respectively. When the ECLIA was used as the gold standard, the sensitivities of the RDT, NAT and RDT and NAT in parallel were 80.7% (95% CI: 73.9~86.4), 86.7% (95% CI: 80.6~91.5) and 93.4% (95% CI: 88.5~96.6), respectively, and the sensitivity of the RDT and NAT in parallel was greater than that of the RDT alone (p < 0.001). The parallel RDT and NAT revealed an additional cost‒benefit ratio (ACBR) < 1 for males and individuals aged over 30 years, which indicated that switching from the RDT screening strategy to the RDT and NAT in parallel is more cost effective. Adults aged over 30 years are the main population with hepatitis B infection in Guangdong Province, China, whose prevalence of HBsAg-ECLIA was 11.18%. Single RDT screening is prone to miss individuals with low levels of HBsAg. It is recommended to implement an RDT and NAT in parallel for individuals older than 30 years. Full article

In this study, we developed a multiplex one-tube nested real-time fluorescent quantitative PCR (mONRT-PCR) assay integrated with a portable, fully automated nucleic acid point-of-care testing (POCT) platform for the detection of Haemophilus influenzae (H. influenzae), Listeria monocytogenes (L. monocytogenes), and Cryptococcus neoformans (C. neoformans) in cerebrospinal fluid (CSF). The assay enables nested amplification within a closed system using conventional primers and probes, thereby reducing operational complexity and minimizing contamination risk. Analytical evaluation demonstrated limits of detection of 10⁰ copies/μL for H. influenzae and L. monocytogenes, and 10¹ copies/μL for C. neoformans using recombinant plasmids, as well as 10⁻⁷ to 10⁻⁶ ng/μL using genomic DNA. No cross-reactivity was observed when tested against a panel of 17 common non-target microorganisms encountered in clinical microbiology laboratories. In simulated CSF samples, the assay maintained detectable amplification at low pathogen concentrations. When implemented on the POCT platform, detection limits reached 5, 10, and 50 CFU/mL for the three pathogens, respectively. Clinical evaluation using 43 CSF samples showed almost perfect agreement with conventional qPCR (κ = 0.861, p < 0.001). Notably, additional C. neoformans detections were observed by mONRT-PCR-POCT compared with qPCR, suggesting improved sensitivity under clinical conditions. The assay yielded results within approximately 1 h and 47 min. These findings indicate that the proposed assay provides a rapid, sensitive, and integrated approach for meningitis pathogen detection, while maintaining a practical balance between analytical performance and operational simplicity. Further validation in larger cohorts is warranted. Full article

Antimicrobial resistance (AMR) is an ever-growing threat to global healthcare. It is largely driven by delayed or inadequate pathogen identification and antimicrobial susceptibility testing in routine clinical workflows. By the time the clinician receives results to guide treatment from traditional culture-based diagnostics, several days may have elapsed, leading to the use and potential over-prescription of broad-spectrum antibiotics and the development of resistant pathogens. A rapid and clinically actionable diagnostic approach at the clinical point of care (POC) may help address this gap. This review examines current and emerging POC diagnostic technologies for AMR and outlines the fundamental principles and mechanistic classifications of POC diagnostic technologies. These include phenotypic, genotypic, immunological, and biosensor-based approaches. A critical overview of key technological platforms, including rapid phenotypic antimicrobial susceptibility testing (AST), microfluidics and isothermal nucleic acid amplification (e.g., LAMP and RPA), CRISPR-based diagnostics, nanomaterial-enhanced biosensors, and mobile-integrated systems is provided. The impact of POC diagnostics on antimicrobial stewardship, time to appropriate therapy, and patient outcomes in primary care settings, hospitals, intensive care units, and resource-limited settings is presented and discussed. In addition to clinical implementation challenges, this review considers the issues of analytical performance, workflow, regulatory pathways, cost, and implementation readiness. In addition, it outlines key trends regarding digital integration, surveillance, workforce training, and policy frameworks. Overall, the review outlines the role of POC diagnostics in enhancing antimicrobial response surveillance and the global fight against AMR. Among emerging platforms, rapid phenotypic AST, microfluidic and isothermal-based assays, CRISPR-based diagnostics, and integrated biosensor systems show the greatest potential for near-term clinical impact; however, widespread implementation remains constrained by challenges related to clinical validation, cost, workflow integration, and alignment with antimicrobial stewardship frameworks. Full article

Background: Molecular syndromic stool panels are increasingly used in paediatric diarrheal syndromes; however, interpretation of Clostridioides difficile (C. difficile) detection remains challenging because colonisation is common in younger children. We aimed to assess the frequency of C. difficile detection using a syndromic gastrointestinal panel in a paediatric tertiary-care centre and to describe the subsequent microbiological work-up and CDI-directed treatment. Methods: We conducted a retrospective single-centre study of all BioFire FilmArray Gastrointestinal (GI) panels performed at San Marco Hospital (University Hospital “G. Rodolico-San Marco”, Catania, Italy) from 1 January 2023 to 31 December 2025. Only the first C. difficile-positive result per patient was included; repeat positives within 30 days were excluded. Index-positive episodes were stratified by age (<1 year, 1 to <2 years, and ≥2 years). Data collected included co-detected pathogens, toxin A/B enzyme immunoassay (EIA) results, GeneXpert PCR findings, and CDI-directed therapy. Results: Among the 714 GI panels performed during the study period, 112 (15.7%) were positive for C. difficile. After exclusion of repeat positives, 91 index-positive episodes were analysed. Median age was 1.0 years (IQR 0.75–4.0), and 48/91 cases (52.7%) occurred in children younger than two years. Toxin A/B EIA was positive in 11/82 tested episodes (13.4%), whereas GeneXpert tcdB was positive in 75/84 episodes (89.3%). Co-detection of at least one additional enteric pathogen occurred in 40/91 cases (44.0%). CDI-directed therapy was administered in 9/91 episodes (9.9%), mainly in children aged ≥2 years. Conclusions: Detection of C. difficile by syndromic molecular panels was relatively frequent in our paediatric cohort but rarely associated with toxin positivity or the need for specific treatment. These findings suggest that many positive Nucleic Acid Amplification Test (NAAT) results may represent colonisation rather than true infection, particularly in younger children. Careful clinical interpretation of syndromic panel results is therefore essential to avoid overdiagnosis and unnecessary antimicrobial therapy. Full article

Cancer remains a major global health challenge, and the limitations of conventional therapies have intensified interest in treatment strategies that combine improved selectivity with reduced systemic toxicity. Photothermal therapy and photodynamic therapy have emerged as minimally invasive approaches capable of achieving spatiotemporally controlled tumour ablation. In this context, molybdenum disulfide (MoS₂), a transition metal dichalcogenide with strong near-infrared absorption, high photothermal conversion efficiency, and versatile surface chemistry, has gained increasing attention as a multifunctional platform for drug delivery and light-triggered cancer therapy. This review examines recent advances in engineered MoS₂ nanoplatforms for drug-enhanced cancer phototherapy, with emphasis on how surface design and therapeutic cargoes mechanistically amplify light-triggered tumour killing. Approaches such as polymer coatings, biomimetic membranes, targeting ligands, chemotherapeutic agents, nucleic acids, and photosensitisers have been explored to improve colloidal stability, tumour targeting, immune evasion, and stimulus-responsive drug release, while also adding complementary cytotoxic pathways such as chemotherapy, ROS generation, or gene silencing. Available in vitro and in vivo studies indicate that these systems generally exhibit favourable short-term biocompatibility under the tested conditions and can produce significant antitumour effects following irradiation. The review also discusses key biological barriers and translational challenges, including biodistribution, long-term safety, reproducibility, and regulatory considerations, highlighting opportunities for the development of clinically viable MoS₂-based phototherapeutic platforms. Full article

The translation of nucleic acid amplification into practical point-of-care and biosensor-integrated diagnostics is still significantly impeded by the necessity for rapid sample preparation. For this reason, a broad comparison of seven commercially available kits for DNA/RNA extraction containing their temperature-related adjustments was performed. Extracts isolated from SARS-CoV-2-positive nasopharyngeal swabs, viral stocks, as well as laboratory-prepared suspensions of clinically relevant Gram-positive and Gram-negative bacteria were evaluated by recombinase polymerase amplification (RPA) and real-time PCR. In addition, the impact of transport media for SARS-CoV-2 samples was investigated. Extraction performance varied markedly according to the kit, pathogen, sample background. For SARS-CoV-2, rapid extraction was more effective for samples collected in viral transport medium than in inactivation buffer. Across bacterial targets, performance was species dependent, highlighting substantial differences in compatibility between simplified extraction workflows and downstream amplification. Among the rapid methods tested, a simplified QuickExtract protocol (95 °C, 5 min) provided the most consistent overall results, although it did not uniformly match the reference silica-based method for all targets. In conclusion, these results demonstrate that rapid nucleic acid extraction must be thoroughly evaluated as an essential element of the entire sample-to-answer workflow, rather than being chosen as a standalone preprocessing step for point-of-care molecular diagnostics. Full article

Human papillomavirus (HPV)-driven oropharyngeal squamous cell carcinoma (OPSCC) has emerged as a biologically distinct entity, typically affecting younger, non-smoking patients and showing improved survival compared to HPV-negative tumors. Accurate HPV status determination is essential for correct staging, prognostic assessment, and treatment de-escalation. Despite advances, substantial variability persists among diagnostic methods and clinical workflows. A narrative review of PubMed, Scopus, and Web of Science databases was conducted up to July 2025. Studies addressing HPV detection techniques in OPSCC—including p16^INK4a^ immunohistochemistry (IHC), HPV DNA and RNA assays, liquid biopsy approaches, and computational surrogates—were critically analyzed regarding diagnostic accuracy, clinical applicability, and emerging innovations. Tissue-based assays remain the diagnostic reference standard. p16 IHC provides high sensitivity but limited specificity and should be confirmed with nucleic acid-based methods such as DNA PCR, in situ hybridization (ISH), or E6/E7 mRNA detection. Combined or “orthogonal” testing minimizes discordance and refines risk stratification. Liquid biopsy detection of circulating HPV DNA using droplet digital PCR or next-generation sequencing has shown high sensitivity and specificity in cohorts of patients with HPV-associated OPSCC, supporting its potential role as a complementary biomarker for treatment monitoring and surveillance. However, circulating HPV DNA alone does not unequivocally identify the anatomic source of HPV DNA and should be interpreted together with clinical, radiologic, and tissue-based findings. Oral rinse and saliva assays show moderate diagnostic performance, while artificial intelligence-based radiomic and histopathologic models are emerging as complementary tools. Reliable HPV attribution in OPSCC requires a multimodal diagnostic strategy integrating p16 IHC, molecular confirmation, and ctHPV-DNA monitoring. Methodological standardization and prospective validation are essential to implement precision-guided, cost-effective workflows in routine clinical practice. Full article

Background: Schistosoma spp. are trematodes occurring in tropical endemic areas but can be imported to non-endemic regions as causes of travel-associated infections. In this study, two pan-trematode-specific real-time PCR assays were evaluated for their diagnostic sensitivity in detecting Schistosoma spp. DNA in diagnostic human samples. Methods: Two previously described pan-trematode-specific real-time PCR assays were comparatively assessed using diagnostic samples containing DNA of either the S. haematobium complex or the S. mansoni complex, as confirmed by Schistosoma species complex-specific real-time PCR. Results: Out of a total of 655 samples containing Schistosoma spp. DNA, positive signals in at least one of the two pan-trematode real-time PCR assays were recorded for 17 (2.6%) nucleic acid extractions. Although sensitivity was in the >90% range for stool samples, only a few individual blood plasma and serum samples, and none of the Schistosoma spp. DNA-containing tissue or urine samples, tested positive by pan-trematode PCR. The lower sensitivity of pan-trematode PCR compared with Schistosoma spp.-specific PCR was semi-quantitatively confirmed by higher cycle threshold (Ct) values in the former. When comparing samples with concordant versus discordant positive results for Schistosoma spp.-specific and pan-trematode PCR, Ct values of the Schistosoma spp.-specific PCR were lower in concordantly positive samples than in discordantly positive samples. Conclusions: While the assessed pan-trematode PCR assays showed insufficient sensitivity as screening tools for blood plasma, blood serum, tissue, and urine samples from individuals with suspected schistosomiasis, they were sufficiently sensitive when applied to stool samples, in which substantial amounts of target DNA, as indicated by low Ct values in the Schistosoma species complex-specific real-time PCR assays, can be expected. For screening for Schistosoma spp. DNA in sample materials other than stool, the use of highly sensitive target-specific PCR remains necessary. Full article

Background: Anti-N-methyl-D-aspartate receptor (anti-NMDAR) encephalitis is an autoimmune disorder frequently associated with ovarian teratomas in young women. Although infectious triggers have been proposed to contribute to immune activation, direct evidence linking viral presence within tumor tissue to disease pathogenesis remains limited. Case Presentation: An 18-year-old woman presented with acute neuropsychiatric symptoms, fever, gastrointestinal prodrome, and rapidly progressive behavioral disturbance progressing to encephalopathy. Cerebrospinal fluid and blood test results, together with clinical features, supported the diagnosis of anti-NMDAR encephalitis. Imaging identified an ovarian mass, and surgical resection was performed. Histopathology confirmed a mature teratoma containing neuroglial elements. Molecular analysis detected parvovirus B19 DNA within the resected teratomatous tissue. No systemic viremia or active central nervous system viral infection was identified. The patient received immunotherapy combined with tumor removal, with subsequent clinical improvement. Discussion: Ovarian teratomas remain a critical etiologic factor in anti-NMDAR encephalitis and mandate prompt surgical management. Detection of B19 viral DNA within teratomatous neuroglial tissue raises the hypothesis that viral persistence could enhance local immune activation and autoantibody generation. However, in this case polymerase chain reaction positivity does not indicate active infection, and the biological significance of this finding remains uncertain. Conclusions: This case documents rare detection of B19V DNA within an ovarian teratomatous tissue in anti-NMDAR encephalitis. The observation is hypothesis-generating rather than causal; established management priorities remain immunotherapy and tumor resection, and viral nucleic acid detection should be interpreted within the broader clinical context. Full article

The development of rapid and sensitive point-of-care nucleic acid tests benefits from robust synthetic recognition elements. Here, a biotin-specific molecularly imprinted polymer (MIP) was synthesized using an optimized protocol and integrated as a biomimetic test zone into two paper-based formats: nucleic acid vertical flow (NAVF) and nucleic acid lateral flow (NALF). Both platforms were evaluated for the detection of double-tagged PCR amplicons from Escherichia coli. NAVF enabled a 3 min visual readout with an LOD of 1.00 × 10⁻² ng mL⁻¹. NALF provided a total assay time of <15 min and achieved a visual LOD of 3.17 × 10⁻² ng mL⁻¹. Overall, the results demonstrate the versatility of biotin-MIPs as stable synthetic receptors for rapid, low-cost paper-based nucleic acid assays, with NAVF prioritizing speed and design flexibility and NALF prioritizing higher analytical sensitivity. Full article

Based on the follow-up of patients who recovered from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, several reports of people who re-tested positive have been described. This may result from viral reactivation, true reinfection, superinfection, or an initial infection by more than one virus (multiple infection). These scenarios can only be correctly distinguished through viral quasispecies analysis. Herein, 26 cancer patients under extended follow-up for SARS-CoV-2 infection were submitted to multiple longitudinal analyses through nucleic acid isolation, PCR amplification and high-throughput sequencing. SARS-CoV-2 classification and the definition of cases as persistent or repeated infections were based on phylogenetic reconstruction. Supported by their viral complete genomes and intrahost quasispecies over time, the different scenarios were identified. Nine confirmed and 12 plausible persistence cases were identified. Virus evolution dynamics in the intrahost population from patients with persistent infection was shown for the first time. Regarding reinfection, three confirmed and two plausible cases were identified, including one case of multiple infection. Altogether, this is the first study that analyzes the plethora of SARS-CoV-2 within-host minor variants and describes reinfections, multiple infections and viral evolution across time in cancer patients, contributing to the understanding of SARS-CoV-2’s within-host population dynamics in the natural history of COVID-19. Full article

Background/Objectives: Microbiological confirmation of suspected complicated urinary tract infections (cUTIs) is challenging, particularly in patients previously exposed to antibiotics or when fastidious organisms are involved. Molecular assays detect microbial nucleic acids independently of bacterial viability and may therefore yield results that differ from conventional culture. This study compared microorganism detection patterns and inter-method agreement between multiplex real-time PCR (mPCR), standard urine culture SUC, and rapid nephelometric screening (Uroquattro HB&L). Methods: In a prospective single-centre study, urine samples from 72 hospitalized patients with clinical suspicion of cUTIs were analyzed using SUC, mPCR (Novaplex™ UTI panel), and the Uroquattro system. Detection rates were calculated for each method. Agreement between paired methods was evaluated using Cohen’s kappa, and paired differences in detection were assessed using McNemar’s and Cochran’s Q tests. Results: mPCR detected microorganisms in 83.3% of samples, compared with 47.2% for SUC and 42.6% for Uroquattro. Agreement between mPCR and SUC was fair (κ = 0.26), whereas SUC and Uroquattro demonstrated high concordance. mPCR identified a broader spectrum of organisms, including fastidious and polymicrobial findings that were not recovered by culture. Correlation between PCR cycle threshold values and colony counts was weak and not statistically significant. Conclusions: mPCR demonstrated a substantially higher microorganism detection frequency than culture-based methods; however, the assays target different biological characteristics, highlight bacterial nucleic acid versus viable growth, and should be interpreted as complementary rather than interchangeable. Conventional culture remains necessary for antimicrobial susceptibility testing and clinical decision-making. Further studies incorporating clinical outcome-based reference standards are required to determine the clinical relevance of molecular detection in cUTIs. Full article

Background: low-quantity or degraded samples are often studied in forensic genetics. Therefore, it is important to efficiently obtain all the available DNA from the biological sample analyzed to provide the most reliable results. This is particularly challenging in bone marrow processing due to its hydrophobic molecular structure, as for other lipid-rich tissues, especially if rancid. In fact, during adipose tissue decomposition, the putrefaction of fatty acids can in some instances give a compact cerous consistency to the lipidic tissue, hardly susceptible to the nucleic acid extraction mechanisms. According to environmental circumstances, this condition is notably observable in submerged bodies or in putrefied bone marrow. Thus, this study is focused on developing an optimized nucleic acids extraction protocol for putrefied bone marrow. Methods: genetic analyses were performed on putrefied yellow bone marrow collected from 20 human femora recovered from bodies in different decomposition stages. The optimized method was developed by integrating additional steps, reagents and time intervals on a silica-based column commercial kit. This strategy was compared in DNA yield to a standard extraction protocol, represented by the same commercial kit, but following the manufacturer’s directions. Both these strategies were tested in nucleic acid isolation efficiency by performing DNA typing, including real-time PCR quantification, Short Tandem Repeats (STR) amplification and fragments analysis steps. The analytical parameters evaluated were allele count, DNA concentration (ng/µL) and Degradation Index (DI). Results: for allele count and DNA concentration parameters, the optimized protocol showed clear and significant qualitative and quantitative improvements compared with the standard protocol, supporting its potential applicability in forensic casework and laying the foundation for future studies. Conclusions: prior to appropriate laboratory internal validation, the optimized protocol can be used for tough lipid-rich tissues processing without the need to purchase a dedicated system and using a same commercial kit routinely adopted for other forensic genetics matrices. Full article

Background: Malignant pleural effusion (MPE) represents a frequent and clinically challenging manifestation of advanced malignancy, particularly in metastatic non-small cell lung cancer (NSCLC). Its management requires integration of diagnostic imaging, symptom-directed therapeutic strategies, and, increasingly, molecular profiling technologies. Recent advancements in this field based on liquid medium (so-called liquid biopsy) have achieved a significant increase in sensitivity, enhancing our ability to investigate biofluids and suggesting their potential integration into standard diagnostic practices, far beyond the canonical plasma biopsies. Fluid obtained from MPE after cytological sample centrifugation is rich in cell-free DNA and less susceptible to nucleic acid degradation during processing, improving overall diagnostic accuracy. Methods: This narrative review summarizes current evidence on the clinical management of malignant pleural effusions in patients with metastatic NSCLC, integrating imaging, procedural management, and molecular profiling from a multidisciplinary tumor board perspective. The primary objective was to synthesize contemporary knowledge with particular attention to the feasibility, reliability, and reproducibility of pleural fluid-based molecular testing. Results: MPE poses diagnostic and therapeutic challenges for all members of the multidisciplinary tumor board, traditionally associated with an adverse prognosis. However, recent advances in cytopathology, histopathology, and liquid-based techniques demonstrate that MPE could be an important source of prognostic or predictive information. At the same time, optimal patient management requires careful integration of imaging findings and procedural strategies (such as pleurodesis or indwelling pleural catheters) with individualized systemic therapy selection. Cell-free DNA in pleural effusions is a promising field of exploration and study, potentially suitable for future guideline implementation, after validation in adequately powered studies, contributing to improving patient management, particularly useful in fragile subsets. Conclusions: The management of MPE in advanced NSCLC is evolving toward a multidisciplinary, precision-oriented model that integrates clinical evaluation, imaging, procedural interventions, and molecular testing. Liquid biopsy technology has gained enough analytical robustness and clinical feasibility to be a useful tool in routine analysis. Biofluid-based molecular testing may have outstanding potential, contributing to improving patient management, avoiding repetitive procedures, and optimizing the overall efficiency and cost-effectiveness of diagnostic practices. Moreover, collaborative projects among different specialties help in consolidating trust in the tumor board decision-making process. Full article