Search Results (75)

Background: Molecular diagnostics may detect several respiratory pathogens simultaneously with rapid turnaround times. The aim of this study was to determine the frequency and distribution of respiratory pathogens among symptomatic outpatients. Methods: All outpatients presented for testing due to suspected acute respiratory infection between 1 January and 31 December 2024 to the Teaching Institute for Public Health of Split-Dalmatia County, Croatia, and multiplex real-time PCRs for 13 respiratory pathogens were included. Results: Out of 15,437 analyzed panels, 8878 (57.5%) were positive. Single-pathogen infections dominated (82.6%), while co-infections were recorded in 17.4% of panels; therefore, a total of 10,546 individual pathogens were detected, which were mostly viruses (87.0%). The following distribution of pathogens was observed: rhinovirus/enterovirus in 38.9% of positive results, influenza A virus in 14.5%, SARS-CoV-2 in 9.5%, parainfluenza virus in 7.9%, respiratory syncytial virus in 7.3%, Mycoplasma pneumoniae in 4.9%, Bordetella pertussis in 4.6%, human metapneumovirus in 4.2%, adenovirus in 3.4%, Chlamydia pneumoniae in 3.4%, influenza B virus in 1.3%, Bordetella parapertussis in 0.1% and Legionella pneumophila had one positive result. The first trimester of the year had the highest number of positive test panels (47.0%). Conclusions: Our study demonstrates a predominance of viral pathogens across all age groups and seasons, further supporting guideline-based practice and highlighting the importance of confirming bacterial infection before initiating antibiotic therapy. This insight into the post-pandemic circulation of respiratory pathogens may help inform public health strategies, including improved surveillance, anticipation of seasonal outbreaks, and targeted interventions, thereby supporting future pandemic preparedness and mitigation efforts. Full article

Poultry disease outbreaks are frequently multifactorial, involving complex interactions between infectious agents and environmental stressors. This report describes a case of avian influenza (AI) co-infection in a commercial broiler farm located in Majalengka, West Java, Indonesia, where a total of 11,000 birds exhibited increased mortality during the grow-out period. Diagnostic investigations included pathological examination, serological testing—such as hemagglutination inhibition (HI) assays for AI H5 and H9, enzyme-linked immunosorbent assay (ELISA) for infectious bronchitis, and rapid testing for Mycoplasma gallisepticum—Polymerase Chain Reaction (PCR) analysis, fecal flotation for coccidiosis, and an environmental assessment measuring ammonia levels, humidity, and heat stress index. Pathological findings revealed tracheitis, airsacculitis, thymitis, bursitis, perihepatitis, ascites, petechial hemorrhages, nephromegaly, and enteritis, indicating a complex disease process. Serological results demonstrated exposure to AI H9 and H5, with differing infection dynamics, while the uneven distribution of infectious bronchitis antibodies suggested field challenges. Additionally, partial exposure to Mycoplasma gallisepticum was observed. PCR results were negative at the time of sampling, consistent with post-infection phases. Environmental evaluation revealed elevated ammonia levels, excessive humidity, and a high heat stress index. In conclusion, the increased mortality was associated with avian influenza co-infection, complicated by secondary infections and adverse environmental conditions. Full article

Background. Overcrowding in emergency departments (EDs), particularly pediatric emergency departments (PEDs), remains a significant challenge that affects patient outcomes and the efficiency of healthcare. Rapid diagnostic tests (RDTs) for respiratory viruses could be a promising tool for improving patient management by enabling prompt etiological diagnoses. This study investigated whether positive RDT results for influenza or adenovirus were associated with differences in length of stay (LOS) in a tertiary PED during epidemic seasons. Methods. A retrospective cohort study was conducted at IRCCS Istituto Giannina Gaslini, Genoa, Italy, over two epidemic seasons (December–February, 2023–2025). All consecutive pediatric patients presenting with fever and respiratory symptoms who underwent rapid diagnostic testing for influenza and/or adenovirus during two epidemic seasons were included. LOS was assessed as the time from triage to discharge (TTD) and from physician assignment to discharge (ATD). Patients were stratified by positive versus negative RDT results. Analyses between groups used the Mann–Whitney U-test for continuous variables and chi-square or Fisher’s exact test for categorical variables. A two-tailed p-value < 0.05 was considered significant. Results. Of the 1238 patients analyzed, the median age was 3.3 years (IQR 1.4–7.2), with male predominance (58.1%). A total of 330 patients (26.6%) tested positive. Compared with negative results, positive RDTs were associated with shorter median TTD (217.0 vs. 239.0 min, p < 0.001) and ATD (66.0 vs. 148.5 min, p < 0.001), which was consistent in both the influenza and adenovirus subgroups. No significant difference in 72 h readmission rates was observed between groups. Conclusions. Among children tested with RDTs for influenza and adenovirus, positive results were associated with reduced PED LOS without increasing early return visits. While these findings suggest a potential role in supporting patient flow, conclusions regarding the broader impact on PED overcrowding should be drawn with caution. Further prospective studies, including non-tested controls and additional viral targets, are required. Full article

Molecular point-of-care testing (POCT) for respiratory infections has undergone remarkable advancement over the past two decades, driven by technological innovation and urgent clinical needs highlighted by the COVID-19 pandemic. This comprehensive systematic review was conducted following PRISMA 2020 guidelines, synthesizing evidence from 254 peer-reviewed studies published between 2006 and 2026, with detailed analysis of the 30 most relevant papers selected through a rigorous four-stage screening process. The review examines the evolution of molecular POCT technologies, including reverse transcription polymerase chain reaction (RT-PCR), loop-mediated isothermal amplification (LAMP), recombinase polymerase amplification (RPA), and CRISPR-based detection systems. Key findings demonstrate that modern molecular POCT platforms achieve diagnostic performance comparable to laboratory-based testing, with sensitivities ranging from 88% to 100% and specificities from 98% to 100%, while delivering results in 15 to 80 min. These technologies enable rapid, accurate detection of major respiratory pathogens, including SARS-CoV-2, influenza A/B, respiratory syncytial virus (RSV), and atypical bacteria. The integration of microfluidic systems, portable devices, and smartphone-based analysis has expanded access to testing in resource-limited settings, emergency departments, and wearable platforms. This review provides critical insights for clinicians, researchers, and policymakers regarding the current state, clinical applications, and future directions of molecular POCT for respiratory infections. Full article

Background/Objectives: Next to malaria, respiratory viruses, particularly respiratory syncytial virus (RSV), are responsible for the hospitalization and death of thousands of young children each year in sub-Saharan Africa. During peak seasons, conducting separate tests is time-consuming and distressing. This underscores the need for efficient, rapid multiplexed diagnostic tools. This study aimed to evaluate the clinical performance of a lateral flow assay (LFA) based antigen combo rapid diagnostic test (ML Ag Combo RDT, manufactured by MobiLab) that detects RSV, influenza viruses A and B (Flu A/B), and SARS-CoV-2. Methods: The Allplex panel 1 rRT-qPCR assay was used as a reference assay to evaluate the clinical performance of the LFA Ag Combo RDT in pediatric hospital settings. It was performed using 470 nasopharyngeal swab (NPS) specimens from hospitalized children under two years of age with respiratory symptoms. Results: Based on the comparative analysis of the testing results for 470 NPS, the ML Ag Combo RDT demonstrated high sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of 90.06%, 98.38%, 93.67, and 97.39% for RSV, and 30%, 100%, 100%, and 95.43 for Flu A/B, respectively. Agreement with the Allplex panle1 1 rRT-qPCR was strong (κ = 0.90 for RSV) and moderate (κ = 0.45 for Flu A/B), with overall accuracies of 96.63% for RSV and 95.5 for Flu A/B. This was further supported by ROC analysis for aggregated data (RSV and, Flu A/B) with an AUC value of 0.925. As expected, in samples with high viral loads (Ct < 20), the Ag Combo RDT achieved 100% sensitivity for RSV and Flu A/B. Sensitivity declined slightly at lower viral loads (Ct > 35). Conclusions: The ML Ag Combo RDT demonstrates high specificity and diagnostic accuracy for the detection of RSV and Flu A/B in pediatric hospital settings where timely diagnosis is critical. Full article

Loop-mediated isothermal amplification (LAMP) is an innovative nucleic acid amplification technique that operates under isothermal conditions and is distinguished by its high analytical efficiency, cost-effectiveness, and operational simplicity. Unlike conventional molecular assays, LAMP does not require sophisticated instrumentation or highly specialized personnel, rendering it particularly suitable for diagnostic deployment in resource-limited settings. Reaction outcomes are typically determined through direct visual inspection, often via colorimetric readouts, further enhancing its applicability in decentralized and point-of-care contexts. Owing to these attributes, LAMP has emerged as a valuable tool for the diagnosis of infectious diseases, particularly in regions with constrained laboratory infrastructure. Its affordability, rapid turnaround time, and ease of implementation support large-scale testing during public health emergencies, including epidemics and outbreaks, thereby contributing to the reduction in disease burden. Timely and accurate pathogen detection using LAMP can substantially strengthen public health responses aimed at controlling and mitigating viral transmission. This review provides an overview of the LAMP methodology, with an emphasis on its application in the detection of viral pathogens with epidemic and pandemic potential. Dengue virus and influenza virus are discussed as representative model infections to illustrate the diagnostic performance and practical advantages of LAMP-based assays. In addition, we explore current challenges and future perspectives for the implementation of LAMP in resource-limited settings, highlighting the need for continued technological refinement and contextual adaptation to maximize its impact on global health initiatives. Full article

Avian influenza (AI), particularly highly pathogenic avian influenza (HPAI), represents a serious and growing threat to global poultry production, international trade, and human health security. Control of AI is complicated by the high evolutionary rate of influenza A viruses, which drives antigenic diversity and ongoing emergence of novel strains. Effective surveillance and disease management therefore depend on timely and accurate diagnostics. While conventional methods—including virus isolation, reverse transcription-quantitative polymerase chain reaction (RT-qPCR), and enzyme-linked immunosorbent assays (ELISAs)—remain effective and widely used, they are limited by long turnaround times, the need for specialized equipment, and reliance on highly trained personnel. In addition, strict state and federal regulatory requirements restrict testing to a limited number of authorized laboratories. Although these regulations are essential for maintaining diagnostic accuracy and quality assurance, they place substantial strain on laboratory capacity during outbreaks and delay actionable results. The need for rapid, on-site decision making has driven interest in alternative diagnostic approaches, including biosensor technologies. A major limitation of current diagnostic strategies is the lack of robust DIVA (Differentiating Infected from Vaccinated Animals) capability. In countries such as the United States, where poultry vaccination against AI is not routinely practiced, the absence of DIVA-compatible diagnostics has hindered adoption of vaccination as a disease management tool, as seropositive birds and products face significant trade restrictions. Biosensor platforms capable of enabling DIVA strategies offer a potential pathway to support vaccination while preserving surveillance integrity. This review examines the current landscape of AI and HPAI diagnostics, emphasizing the limitations of traditional approaches and the opportunities presented by biosensor platforms. We evaluate electrochemical, optical, piezoelectric, and nucleic-acid-based biosensors, with particular attention to biorecognition strategies, performance metrics, field deployability, and applications supporting subtype discrimination, DIVA implementation, and One Health surveillance. Full article

Background: Respiratory tract infections (RTIs) represent one of the most prevalent reasons for visits to Pediatric Emergency Departments (PEDs). Because viral and bacterial presentations frequently overlap, a substantial proportion of antibiotic prescriptions in pediatric acute care are potentially unnecessary, contributing to antimicrobial resistance. Rapid Diagnostic Tests (RDTs) for respiratory viruses have been suggested as tools to enhance diagnostic precision and support antimicrobial stewardship. However, evidence regarding their real-world impact in pediatric emergency settings is limited. Objectives: This study aimed to assess the association between point-of-care RDT results and antibiotic management in a tertiary PED, focusing on both the discontinuation of antibiotics in children already receiving treatment and the avoidance of new antibiotic prescriptions in untreated children. The secondary objective was to evaluate the short-term safety through 72-h return visits. Methods: A retrospective cohort study was conducted at a tertiary PED during two epidemic seasons (December–February 2023–2024 and 2024–2025). Children aged <18 years who underwent RDTs for febrile respiratory illnesses were included. Patients were stratified based on whether they were already receiving antibiotic therapy at presentation. The primary outcomes were antibiotic discontinuation among treated patients and initiation among untreated patients. Unplanned return visits to the PED within 72-h post-discharge were used as a pragmatic short-term safety outcome to capture early clinical deterioration. RDTs (SD Biosensor Standard F Antigen) were performed at the bedside with a turnaround time of 10–15 min. Results: A total of 1238 children were included, of whom 330 (26.6%) tested positive for influenza and/or adenovirus. Among the 234 children already receiving antibiotics, discontinuation was significantly more frequent in the RDT-positive group (p < 0.001; OR 0.044). Among the 1004 untreated children, antibiotic prescription was significantly lower in the positive group than in the negative group (p < 0.001; OR 0.097). Return visits within 72-h did not differ between the groups in either cohort. No invalid tests occurred. Conclusions: Influenza/adenovirus RDT positivity was associated with lower antibiotic initiation among untreated children and higher discontinuation among those already receiving antibiotics, with no differences in 72-h return visits. These findings suggest a potential role for bedside viral testing as a decision-support tool for antibiotic management in the PED. Full article

The overlapping circulation of influenza (Flu), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2; SC2), and respiratory syncytial virus (RSV) continues to challenge clinical laboratories, particularly in settings with limited automation and fragmented healthcare coverage. This study expanded the CDC Flu-SC2 assay by incorporating a laboratory-developed test (LDT) for RSV A/B detection into a fully automated quadruplex RT-qPCR (LDRA) on the Panther Fusion^® Open Access™ system. The design, based on more than 8000 RSV genomic sequences targeting the conserved M gene, achieved optimal amplification efficiencies (97–105%) and full multiplex compatibility. Analytical assessment established limits of detection between 9.6 and 37.8 copies per reaction, absence of cross-reactivity with 30 respiratory pathogens, and inclusivity for 32 viral variants. Commutability and diagnostic performance among the LDRA, CE IVD-marked Allplex™ SARS-CoV-2/FluA/FluB/RSV, and US IVD-marked Panther Fusion^® SARS-CoV-2/Flu A/B/RSV Assays were evaluated using 405 nasopharyngeal UTM-preserved swabs. The LDRA demonstrated excellent concordance (overall agreement ≥ 98%, κ > 0.95), strong diagnostic accuracy, and reliable detection of mixed infections. This quadruplex provides a fully automated, rapid, and accurate solution for the simultaneous detection of influenza A, influenza B, SARS-CoV-2, and RSV viruses, enhancing molecular diagnostic capacity and supporting equitable, timely clinical decision-making in middle-income healthcare systems such as that of the Dominican Republic. Full article

Chicken respiratory diseases represent multifactorial conditions resulting from viral, bacterial, mycoplasmal pathogens, and environmental factors, causing significant economic losses within the poultry industry. A specific respiratory disease characterized by breathing difficulties and bronchial occlusion due to caseous exudates is termed chicken bronchial obstruction. However, the absence of rapid, precise, and highly sensitive diagnostic methods for differentiation of primary respiratory disease pathogens or opportunistic pathogens, including avian influenza virus (AIV), infectious bronchitis virus (IBV), Pseudomonas aeruginosa (P. aeruginosa), and Escherichia coli (E. coli), constitutes a substantial challenge. This study developed a quadruplex droplet digital polymerase chain reaction (ddPCR) method that targeted the HA gene of H9 subtype AIV, the M gene of IBV, the Pal gene of P. aeruginosa, and the UidA gene of E. coli. Following the optimization of annealing temperature, sensitivity, and repeatability, the minimum detectable concentrations were determined as 3.02 copies/μL for the HA gene of H9 subtype AIV, 3.08 copies/μL for the M gene of IBV, 3.19 copies/μL for the Pal gene of P. aeruginosa, 3.39 copies/μL for the UidA gene of E. coli. No cross-reactivity was observed with Newcastle disease virus (NDV), H5 subtype AIV, H7 subtype AIV, fowl adenovirus serotype 4 (FAdV-4), infectious laryngotracheitis virus (ILTV), Avibacterium paragallinarum, Streptococcus, Salmonella, Pasteurella multocida, and Staphylococcus aureus. The method demonstrated excellent repeatability, with a coefficient of variation (CV) below 9%. The 185 clinical samples collected in Hebei Province China are tested by both quadruplex ddPCR and quadruplex qPCR method and the results compared. The sensitivity of the quadruplex ddPCR method (57.30%; 106/185) slightly exceeded that of the quadruplex qPCR method (49.73%; 92/185). Pathogens or opportunistic pathogens positive rates obtained via the quadruplex ddPCR were 40.00% for H9 subtype AIV, 33.51% for IBV, 24.32% for P. aeruginosa, and 27.57% for E. coli. In comparison, the positive rates of H9 subtypes AIV, IBV, P. aeruginosa, and E. coli from the quadruplex qPCR were 36.22%, 30.81%, 21.62%, and 24.32%, respectively. The coincidence rates between the two methods were 96.22% for H9 AIV, 97.30% for IBV, 97.30% for P. aeruginosa, and 96.76% for E. coli. These results demonstrated that the quadruplex ddPCR method represented a highly sensitive, specific, and rapid technique for identifying H9 subtype AIV, IBV, P. aeruginosa, and E. coli. Full article

The widespread circulation of Eurasian avian-like H1N1 (EA H1N1) swine influenza virus poses significant zoonotic and pandemic risks worldwide. However, current diagnostic methods are difficult to deploy in the field, as they generally require specialized laboratory infrastructure and trained personnel. Here, we present a novel dual-signal detection platform that combines reverse transcription recombinase polymerase amplification (RT-RPA) with CRISPR/Cas12a technology for rapid, on-site EA H1N1 detection. We established an integrated one-tube assay by designing and optimizing RT-RPA primers targeting a conserved region of the hemagglutinin (HA) gene, together with engineered CRISPR/Cas12a guide RNAs exhibiting high specificity. The platform incorporates two complementary readout modes: real-time fluorescence monitoring and visual colorimetric detection using a smartphone. The assay shows excellent analytical specificity, with no cross-reactivity observed against other swine influenza virus subtypes or common swine pathogens, (including CSFV, PRRSV, PEDV, PCV, TGEV, and RV). The detection limit is 2 copies/μL, and the entire procedure can be completed within 30 mins using simple portable equipment. When evaluated on 86 clinical samples, the assay demonstrated 94.18% concordance with RT-qPCR. Compared with conventional diagnostic methods, this RT-RPA–CRISPR/Cas12a assay offers greater convenience and cost-effectiveness. Its strong potential for field-based rapid testing underscores promising application prospects in swine influenza surveillance and control programs. Full article

Background: Accurate identification of pneumonia pathogens is critical for guiding appropriate antibiotic therapy and minimizing unnecessary antimicrobial exposure. Bronchoalveolar lavage (BAL) is widely used for pathogen detection but introduces procedural risks. Bronchial washing (BW) is simpler and less invasive, yet evidence for its utility in multiplex PCR diagnostics is limited. Methods: This study includes an evaluation of the clinical utility of the BioFire^® FilmArray^® Pneumonia Panel (FA) using BW specimens via comparison with conventional culture. Between 2022 and 2024, 190 BW specimens were collected from 182 adult patients with suspected pneumonia at Eunpyeong St. Mary’s Hospital, Seoul, Korea. Each specimen was tested simultaneously using FA and conventional culture. Results: The culture positivity rate was 41.6%, whereas FA showed a higher positivity rate of 51.1%. Of all specimens, 52.6% (100/190) were positive in at least one of two methods, and 11.0% (21/190) were FA-positive only. FA detected 72 additional bacterial targets, most commonly H. influenzae, K. pneumoniae, S. aureus, S. agalactiae, and S. pneumoniae. Semi-quantitative results demonstrated a statistically significant moderate correlation with culture (ρ = 0.48, p < 0.001). Eight bacterial targets achieved 100% PPA, and resistance genes were rapidly detected, although some discrepancies with phenotypic antimicrobial susceptibility testing were observed. Several FA-only detections may reflect oropharyngeal colonization rather than true infection. Conclusions: FA testing of BW specimens demonstrated high concordance with culture and provided rapid pathogen and resistance gene detection. BW-based FA testing may serve as a useful diagnostic alternative when BAL is not feasible, although careful interpretation is required to account for potential contamination. Full article

Background: Acute Respiratory Infections are a common reason for Pediatric Emergency Department (PED) visits. Differentiating bacterial and viral infections may be challenging and might result in incorrect antibiotic prescriptions and exacerbation of antimicrobial resistance. This study evaluated the impact of new diagnostic tests in PED. Methods: A retrospective cohort of 4882 acute febrile respiratory infection cases presenting to the PED was analyzed, comparing two periods: Period 1 (October 2016–March 2017, n = 2181) and Period 2 (October 2023–March 2024, n = 2701). During Period 1, Group A Streptococcus and Respiratory Syncytial Virus rapid antigen detection tests were available. During Period 2, new point-of-care tests (POCTs), including rapid C-reactive protein and rapid antigen detection for Influenza A, Influenza B, and SARS-CoV-2, and a multiplex PCR nasal swab, were introduced. Results: In Period 2, antibiotic prescriptions decreased by 28.4%, along with a reduction in broad-spectrum antibiotic use. A significant correlation was observed between reduced antibiotic prescription and the use of new POCTs and multiplex PCR tests. Performance of blood tests and chest radiographs also decreased. Conclusions: Implementing novel diagnostic tests in PED helps clinicians select more appropriate management options with an impact on reduced stress and radiation exposure and antibiotic prescription. Full article

Point-of-care (POC) infectious disease diagnostics are reshaping global health by delivering rapid, decentralized, and clinically actionable results that link bedside testing to population-level surveillance. Valued at approximately USD 53 billion in 2024 and projected to nearly double by 2033, the global POC diagnostics market is driven by infectious disease assays and accelerated by innovations in molecular amplification, biosensors, microfluidics, and artificial intelligence (AI). This review integrates current evidence across technological, clinical, regulatory, and public health domains. Immunoassays remain the backbone of volume deployment, while molecular nucleic acid amplification tests (NAATs) and emerging CRISPR-based platforms achieve laboratory-grade sensitivity at the point of care. AI has transitioned from an experimental tool to an embedded analytical layer that enhances image interpretation, multiplex signal deconvolution, and automated quality control. Rigorous validation, including analytical accuracy, clinical performance in intended-use settings, and usability testing under CLIA guidance, remains central to ensuring reliability in decentralized environments. Regulatory frameworks are adapting in parallel: FDA’s lifecycle oversight of AI-enabled devices, the European IVDR’s expanded evidence requirements, and the WHO Prequalification all emphasize continuous post-market surveillance. From a public health perspective, POC diagnostics have improved early case detection, treatment initiation, and outbreak containment for HIV, tuberculosis, malaria, influenza, RSV, and COVID-19. Yet persistent challenges (including limited harmonization of standards, uneven reimbursement, and scarce real-world data from low- and middle-income countries) continue to constrain equitable adoption. POC infectious disease diagnostics are thus entering a pivotal phase of digitization and regulatory maturity. Addressing remaining gaps in validation, lifecycle monitoring, and implementation equity will determine whether these technologies achieve their full promise as clinical accelerators and as cornerstones of global infectious disease preparedness. Full article

In November 2024, an outbreak of highly pathogenic avian influenza (HPAI) H5N1 was confirmed in backyard poultry in Timiș County, Western Romania. The index cases involved chickens and domestic geese found dead with lesions characteristic of HPAI. Laboratory confirmation was achieved by real-time RT-qPCR targeting the matrix, H5, and N1 genes, followed by virus isolation in embryonated specific-pathogen-free eggs. Sequencing of the hemagglutinin cleavage site revealed the multi-basic motif PLREKRRKR/GLFG, consistent with a highly pathogenic phenotype. To investigate potential viral persistence, tracheal and cloacal swabs were collected from apparently healthy selected backyard poultry (chickens, geese, ducks and pheasants). RNA extraction and RT-qPCR, performed using protocols validated by the European Union Reference Laboratory for Avian Influenza, yielded negative results for all samples. Internal controls confirmed assay reliability, excluding the possibility of PCR inhibition. The investigation confirmed the occurrence of HPAI H5N1 in backyard poultry and demonstrated the absence of detectable viral persistence in surrounding flocks under the tested conditions. These findings highlight the importance of rapid molecular diagnostics, active surveillance, and strict biosecurity in limiting virus spread. Continued monitoring under the One Health framework is essential to mitigate the risk of avian influenza at the human–animal–environment interface. Full article