Multiplex PCR has revolutionized clinical microbiology by enabling syndromic testing, a paradigm shift from traditional pathogen-specific diagnostics. Unlike conventional singleplex PCR, which targets one organism per reaction, multiplex PCR uses multiple primer pairs to simultaneously detect numerous pathogens from a single specimen in one run. This approach begins with clinical syndrome identification—such as fever with cough or acute diarrhea—rather than guessing a specific organism, then tests for all known pathogens causing that syndrome. The workflow streamlines from ordered single tests to comprehensive panel analysis, significantly improving diagnostic efficiency. Most modern syndromic panels employ cartridge-based systems, where specimens are added to sealed cartridges containing all necessary reagents. These automated instruments handle extraction, amplification, and detection, requiring minimal staff time and producing results within approximately one hour. For RNA viruses, reverse transcription converts RNA to DNA before amplification proceeds. The output provides a simple detected or not detected report for each target. This syndromic approach has rapidly proliferated through clinical laboratories because it accelerates diagnosis, reduces testing delays, and enables faster therapeutic intervention by simultaneously screening multiple etiologies from single specimens.

Microfluidic droplets are miniature bioreactors created by dispersing femtolitre-to-nanolitre volumes of one fluid within an immiscible carrier fluid inside microchannels. Each droplet functions as a sealed, independent compartment capable of containing a single cell, enzyme, or bacterium. These droplets are formed within microchannels typically tens to hundreds of micrometres wide, with individual volumes spanning the femtolitre to nanolitre range. A key advantage of this technology is reproducibility: a single microdevice produces millions of near-identical droplets per hour, a capability that distinguishes droplet microfluidics from conventional bulk emulsification. Monodisperse droplets—populations with diameters varying by only a few percent—are critical because droplet volume directly determines reagent concentration, incubation conditions, and signal intensity. Droplet size and frequency depend on flow-rate stability, making pressure-driven flow control preferable to syringe pumps due to faster settling and lower flow-rate variation. The dispersed phase typically contains aqueous buffers with cells or reagents, while the continuous phase consists of an immiscible carrier. This technology unlocks numerous life-science applications by providing unprecedented control over biological compartmentalization.

Loop-mediated isothermal amplification (LAMP) represents an advanced molecular biology technique offering rapid, accurate, and cost-effective diagnosis of infectious diseases, particularly in resource-limited settings lacking PCR infrastructure. The technique amplifies nucleic acids through strand displacement reactions and stem-loop structure formation under isothermal conditions, eliminating the need for thermocyclers. During the COVID-19 pandemic, RT-LAMP assays successfully detected SARS-CoV-2 from saliva or swab samples using simple heat sources with visible color change indicators. While conventional diagnostic methods including culture, microscopy, and biochemical tests remain foundational, they present significant limitations: culture methods are slow, direct microscopy lacks sensitivity, and traditional approaches struggle with mixed infections and morphological variations. Advanced serology and antigen tests, though rapid, may lack specificity. LAMP addresses these critical gaps by providing sensitive and specific pathogen detection at point-of-care facilities, clinics, and field settings. This capability is crucial as diagnostic delays directly impact treatment efficacy and disease severity, while misdiagnosis leads to inappropriate medication and antimicrobial resistance development. LAMP's ability to operate with minimal laboratory infrastructure makes it particularly valuable for global infectious disease control and surveillance.

Recent advances in optical technologies have opened new possibilities for electrophysiological interfacing, offering alternatives to traditional electrically-based methods. Here, we present a flexible optical-electrode ‘optrode’ that employs a passive transduction mechanism to convert biopotentials into quantifiable optical signals. Our design integrates liquid crystals into a multilayered polymeric structure that enables light transmission and reflection while carrying the signal to a photodetection system with a relative responsivity of 13 ± 1%/V and an operating bandwidth of 2.2 ± 0.1 kHz for electrophysiology applications (N = 25 total measurements). Additionally, we demonstrated its cytocompatibility and effectiveness in detecting electrograms from cardiac tissues. This approach addresses major limitations of biomedical optoelectronics, providing label-free and conductive lead-free signal detection while maintaining a soft, tissue-like interface and demonstrating strong potential for integration into advanced electrophysiological platforms.

Viral infectious diseases continue to pose a profound threat to global health security, necessitating a strategic transition from centralized, laboratory-bound diagnostics to scalable and decentralized monitoring systems. This review provides a comprehensive synthesis of the role played by optical biosensors in this transformation, spanning rapid pathogen detection, immune monitoring, and vaccine evaluation. Recent advances in nanotechnology including plasmonic metasurfaces, quantum dots, and graphene-based transducers have empowered the detection of high-risk viruses such as SARS-CoV-2, Ebola, Zika, and Monkeypox with sensitivities frequently comparable to gold-standard molecular diagnostics in controlled laboratory settings. In parallel, the early-stage integration of Artificial Intelligence (AI) and the Internet of Medical Things (IoMT) is facilitating the conversion of passive biosensing components into hybrid optical-digital platforms, paving the way for advanced data processing and personalized immune profiling. Furthermore, we examine the integration of these platforms into point-of-care (POC) diagnostics and longitudinal monitoring, while critically assessing the current translational limitations of wearable bioelectronic interfaces for continuous health surveillance. By addressing existing translational bottlenecks such as scalability, matrix interference, and clinical validation we propose a strategic roadmap for advancing optical biosensors from proof-of-concept prototypes to deployable tools for public health management.

Antimicrobial resistance represents a critical global health threat driven by inappropriate antibiotic use and bacterial adaptability. This comprehensive narrative review examines resistance mechanisms in both Gram-positive and Gram-negative bacteria, with focus on clinically significant ESKAPE pathogens. The analysis covers four principal resistance mechanisms: enzymatic drug inactivation, reduced drug uptake, target site modification, and active efflux, comparing their molecular basis and clinical implications across organism groups. Biofilm formation is discussed as a resistance-amplifying strategy with organism-specific variations in matrix composition. The review evaluates current and emerging diagnostic approaches for AMR detection and explores pharmacokinetic/pharmacodynamic optimization as a resistance-prevention strategy. Notably, lariocidin—a structurally novel ribosome-targeting lasso peptide—is presented as a promising emerging therapeutic scaffold demonstrating broad-spectrum activity against multidrug-resistant pathogens from both bacterial groups while circumventing existing resistance mechanisms. The article addresses particular challenges in pediatric populations, where diagnostic difficulties and empirical prescribing practices amplify resistance impacts. The authors emphasize that combating AMR requires coordinated global efforts integrating surveillance, research, policy-making, and education to preserve antimicrobial treatment efficacy.

Researchers at the University of Texas Medical Branch evaluated the clinical laboratory performance of bioMérieux's VITEK REVEAL® rapid phenotypic antimicrobial susceptibility testing system against the standard MicroScan WalkAway Plus system. The study analyzed 200 positive blood culture bottles containing Gram-negative organisms, assessing categorical and essential agreement rates along with error classifications. VITEK REVEAL achieved essential agreement of at least 90 percent across most antimicrobial-organism combinations evaluated among 2,536 total pairings, with notably lower performance only for amoxicillin/clavulanate and ampicillin/sulbactam. Error rates included 14 very major errors, 16 major errors, and 82 minor errors, with some discrepancies attributed to limited resistant isolates and breakpoint-adjacent minimum inhibitory concentration distributions. Supplemental Etest analysis reduced selected discrepancies. By delivering phenotypic susceptibility results directly from positive blood cultures within eight hours, VITEK REVEAL offers potential for accelerated antimicrobial therapy optimization and stewardship programs in bloodstream infection management. However, the authors acknowledge limitations including reliance on an automated comparator rather than reference broth microdilution and local resistance patterns, recommending additional multicenter studies to establish clinical impact.

Acute respiratory infections remain a leading cause of mortality in children under five, particularly in low- and middle-income countries, with over 650,000 annual deaths globally and highest incidence in Africa and Southeast Asia. Conventional diagnostic methods such as culture and biochemical tests are time-consuming and lack sensitivity, while molecular techniques like PCR require specialized equipment unsuitable for resource-limited settings. To address these limitations, researchers have developed Recombinase Polymerase Amplification (RPA)-ELISA as an alternative diagnostic approach. RPA is an isothermal nucleic acid amplification technique that operates at mild temperatures between 37-42°C and produces results within 20-30 minutes, making it ideal for point-of-care testing. Studies have demonstrated that RPA successfully detects various respiratory pathogens including Streptococcus pneumoniae, Mycobacterium tuberculosis, and respiratory viruses with sensitivity and specificity comparable to traditional PCR methods. This innovation offers a rapid, portable, and cost-effective diagnostic solution for identifying bacterial respiratory infections, enabling timely treatment and infection control in resource-constrained healthcare settings where such tools are critically needed.

CRISPR technology has evolved from a rapid-response tool during the Zika epidemic into a comprehensive platform addressing infectious disease management in two critical ways: enabling point-of-care diagnostics that deliver results within minutes and facilitating environmentally targeted vector control in natural settings. As field applications expand and regulatory frameworks develop, the quality of genome editing reagents emerges as a fundamental determinant of success. Off-target effects and inconsistent editing rates present significant obstacles that can derail diagnostic and vector control initiatives. Achieving reliable, high-precision results requires optimized target selection and specially designed synthetic reagents suitable for scaled deployment. This evolution demonstrates how CRISPR extends beyond basic laboratory research to offer practical solutions for disease prevention and control, with the caveat that technical precision in reagent design directly impacts the effectiveness and reproducibility of real-world applications.

This study examines the clinical drivers, barriers, and system readiness for implementing rapid molecular point-of-care testing (POCT) in respiratory diagnosis across Saudi Arabia. Conducted by a multidisciplinary team of Saudi healthcare professionals, the research addresses the potential of rapid molecular diagnostics to accelerate the identification of respiratory infections, including COVID-19 and influenza. The investigation evaluates the current state of diagnostic infrastructure and identifies key clinical factors motivating the adoption of POCT technology. The study also explores significant implementation barriers within the Saudi healthcare system and assesses organizational readiness for deploying these rapid testing capabilities. The findings are particularly relevant to antimicrobial stewardship initiatives, as faster respiratory pathogen identification can reduce unnecessary antibiotic prescribing and improve patient outcomes. By documenting both the clinical imperatives and systemic challenges, this research provides actionable insights for healthcare policymakers and administrators seeking to modernize diagnostic capabilities in the region and enhance the efficiency of respiratory disease management.

Mycobacterial lung diseases, including tuberculosis and nontuberculous mycobacterial infections, pose a significant global health threat amplified by rising antimicrobial resistance. Traditional drug development approaches using two-dimensional cell cultures and animal models frequently fail to accurately predict drug efficacy and lung penetration, creating critical bottlenecks in therapeutic discovery. To address this translational gap, researchers are adopting New Approach Methodologies (NAMs)—advanced technologies including organoids, air-liquid interface systems, and organ-on-chip platforms that more accurately replicate human lung physiology and host-pathogen interactions. These microphysiological systems enable evaluation of drug distribution, bacterial persistence monitoring, and identification of predictive biomarkers for antibiotic effectiveness. This research initiative establishes an interdisciplinary platform integrating microfluidics, biomaterials, pharmacology, and microbiology to accelerate antibiotic development and optimize treatment strategies. The effort solicits original research and reviews focusing on evaluating novel antibiotics using lung organoids, modeling resistance mechanisms in Mycobacterium tuberculosis and nontuberculous species, identifying efficacy biomarkers through multi-omics approaches, assessing pharmacokinetics in engineered tissue models, and validating NAM-based infection models for high-throughput screening. This comprehensive approach promises to enhance drug discovery efficiency and facilitate development of host-directed therapies against mycobacterial pathogens.

Pseudomonas aeruginosa is a multidrug resistant gram-negative bacterium that poses a significant global health threat, particularly for immunocompromised patients. The organism employs diverse resistance mechanisms including efflux pump overexpression, porin modification, enzymatic inactivation, and target site modifications, alongside phenotypic adaptations such as biofilm and persister cell formation. These complex strategies have driven the emergence of multidrug resistant, extensively drug resistant, and pan drug resistant strains, complicating treatment options. While conventional culture-based diagnostics remain the gold standard, they suffer from delays and limitations in detecting heteroresistance and biofilm-associated tolerance. Recent advances in omics-based approaches—including genomics, epigenomics, transcriptomics, proteomics, lipidomics, metabolomics, and phenomics—offer rapid and precise alternatives for identifying resistant P. aeruginosa. Complementary innovations such as microfluidic lab-on-chip systems and machine learning-driven artificial intelligence further enhance diagnostic capabilities. Integrating multi-omics data with advanced platforms represents a revolutionary strategy for comprehensive, rapid resistance profiling in precision diagnostics. However, standardized protocols, clinical validation, and cost-effective implementation are urgently required to translate this potential into clinical practice and reduce the global antimicrobial resistance burden.

Antimicrobial resistance poses a critical global health crisis, causing over 1.2 million deaths annually with projections reaching 10 million yearly by 2050. Bacteriophage therapy has emerged as a promising alternative to combat multidrug-resistant bacteria, including ESKAPE pathogens, and is advancing through controlled clinical trials supported by the WHO and TATFAR. However, clinical translation remains constrained by the lack of rapid, standardized methods for therapeutic phage selection. Researchers have developed digital phage susceptibility testing (dPhaST), an automated droplet digital PCR workflow that quantifies phage-induced DNA release as a marker of bacterial lysis by targeting conserved 16S rRNA regions. Testing 122 phage-host combinations across six bacterial species, dPhaST achieved 95.9% concordance with traditional spot tests while completing analysis within three hours and resolving weak lytic activities that conventional methods miss. The approach proves robust against phage-encoded nucleases and cross-contamination, capturing defense mechanisms involving CRISPR-Cas and Sir2-HerA systems. This automated quantification method enables rapid, mechanistically informative profiling of phage efficacy across diverse pathogens, significantly advancing phage therapy's clinical potential.

# Summary This review examines optical biosensors as diagnostic tools for viral infectious diseases, providing a comprehensive analysis of their current clinical applications and future potential. Optical biosensors leverage light-based detection mechanisms to identify viral pathogens with high sensitivity and specificity, offering significant advantages over conventional diagnostic methods. The review covers various optical technologies including surface plasmon resonance, fluorescence-based assays, and photonic devices that enable rapid, label-free or minimally invasive virus detection. The analysis encompasses both established viral infections and emerging pathogens, highlighting the role of optical biosensors in point-of-care diagnostics and laboratory settings. Key considerations include assay sensitivity, specificity, cost-effectiveness, and integration with portable platforms. The review emphasizes how these technologies address critical diagnostic challenges such as reducing detection time and improving accessibility in resource-limited settings. Future perspectives focus on nanotechnology integration, multiplexing capabilities, and real-time monitoring applications. Optical biosensors represent a transformative approach to infectious disease diagnosis, with significant implications for pandemic preparedness, early detection strategies, and personalized medicine in virology.

Dr. Zubair Khalid, a veterinarian and virologist, specializes in advancing diagnostic and therapeutic innovations through molecular virology expertise. His research focuses on leveraging CRISPR-Cas12a-based nucleic acid detection systems for rapid pathogen diagnosis, addressing critical gaps in conventional diagnostic methods. This approach represents a significant technological advancement, as CRISPR-Cas12a offers specificity and speed superior to traditional techniques, enabling faster identification of viral and infectious agents in clinical and veterinary settings. Dr. Khalid's work integrates multiple disciplines—conventional virology, molecular biology, and computational approaches—to enhance diagnostic accuracy and accessibility. His expertise in vaccine development and multi-omics analysis further strengthens the translational potential of these innovations. By combining cutting-edge molecular techniques with veterinary science, his research demonstrates substantial promise for improving animal health outcomes and supporting rapid response capabilities during disease outbreaks. This interdisciplinary approach underscores the growing importance of molecular diagnostics in contemporary infectious disease management.

The rapid advancement in personalized healthcare has driven the development of wearable biomedical devices for real-time biomarker monitoring and diagnosis. Traditional invasive blood-based diagnostics are painful and limited to sporadic health snapshots. To address these limitations, microneedle-based sensing platforms have emerged, utilizing interstitial fluid (ISF) as an alternative biofluid for continuous health monitoring in a minimally invasive and painless manner. This review aims to provide a comprehensive overview of microneedle sensor technology, covering microneedle design, fabrication methods, and sensing strategy. Additionally, it explores the integration of monitoring electronics for continuous on-body monitoring. Representative applications of microneedle sensing platforms for both monitoring and therapeutic purposes are introduced, highlighting their potential to revolutionize personalized healthcare. Finally, the review discusses the remaining challenges and future prospects of microneedle technology.

Reliable and continuous access to interstitial fluid (ISF) remains a major challenge for wearable and tissue-embedded biosensing systems. Conventional microneedle-based methods, while widely adopted, often exhibit variable sampling efficiency across different skin types and are prone to performance fluctuations during motion. Herein, we introduce a tissue-embedded thread-based open capillary microfluidics-based sampling that enables minimally invasive, pump-free, and reliable continuous monitoring of small molecules from ISF. The system employs open capillary-driven microfluidic channels in textile threads, which facilitate the partial separation of small molecules from complex biological matrices, mimicking a microdialysis process. The continuous sampling is achieved through evaporation-driven capillary pressure, eliminating the need for an external pump. The experimental results confirm the numerical simulation of diffusion-controlled and capillary-driven transport of analytes in ISF.

Light-enhanced molecular diagnostic technologies are advancing infectious disease detection by overcoming limitations of conventional methods, which often suffer from weak signal strength, slow processing, and difficulty detecting low-abundance targets in complex samples. These innovative approaches leverage plasmonics, photonics, and nano-optical effects to enable enhanced pathogen identification and antimicrobial resistance profiling. Recent studies demonstrate significant progress in fluorescence, chemiluminescence, surface-enhanced Raman scattering (SERS), and photoacoustic applications for sensitive pathogen detection. However, challenges remain in reproducibility, device integration simplification, and real-world clinical validation, particularly for translating laboratory-based optical platforms into point-of-care systems suitable for resource-limited healthcare settings. A comprehensive research initiative aims to strengthen the theoretical and technical foundations of optical signal amplification while fostering clinical implementation of light-enhanced diagnostics. Priority areas include investigating light-matter interactions at nano- and microscales to amplify biological recognition, integrating optical sensing into compact diagnostic platforms, and improving pathogen detection, antimicrobial resistance monitoring, and outbreak response capabilities. The initiative welcomes research on light-induced signal amplification, fluorescence-based platforms, plasmonic materials, photothermal and photoacoustic detection strategies, and microfluidic integration to advance infection diagnostics.

Isothermal nucleic acid amplification technologies (INAATs) represent a transformative alternative to polymerase chain reaction (PCR) for infectious disease diagnostics, addressing critical limitations in resource-limited and decentralized settings. Unlike PCR, which requires thermal cycling equipment and specialized laboratory infrastructure, INAATs including LAMP, RPA, RAA, MIRA, HDA, RCA, SDA, and NASBA enable nucleic acid amplification at constant temperature while maintaining high sensitivity and specificity. These methods support detection of bacterial, viral, fungal, and parasitic pathogens without sophisticated instrumentation. Recent advances in enzyme engineering, assay design, CRISPR-based detection, biosensors, microfluidics, and portable devices have significantly enhanced performance capabilities. This research initiative compiles the latest innovations across methodological developments and clinical applications, including point-of-care diagnostics for outbreak response, clinical diagnosis, and surveillance. The compilation welcomes contributions spanning enzyme optimization, novel amplification chemistries, integration with next-generation detection technologies, and veterinary, environmental, and food safety applications, enabling the transition from conventional laboratory testing to accessible diagnostic platforms where infectious disease burden is greatest.

Researchers have developed a pocket-sized point-of-care testing platform that eliminates the need for electricity, complex laboratory equipment, and skilled technicians. The fully integrated device uses a manual suction pump with a compression spring to operate fluidic functions without external power, achieving high reproducibility with coefficients of variation below 4.5 percent. The platform incorporates a serpentine microchannel design that leverages Dean flow-induced inertial mixing to accelerate molecular aggregation, enabling a wash-free plasmonic assay. Validated with two critical biomarkers—thrombin for cardiovascular and renal diagnostics, and SARS-CoV-2 spike protein for viral screening—the system delivers quantitative results within five minutes with sub-nanomolar detection limits of 0.5 nanomolar for thrombin and 0.4 nanomolar for spike protein. Wireless smartphone connectivity enables immediate data visualization. This innovation addresses critical gaps in decentralized diagnostics, particularly for resource-limited settings and developing countries where conventional laboratory infrastructure is unavailable, while responding to demonstrated pandemic-era demands for rapid, portable diagnostic solutions.