Search Results (35)

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

Sexually transmitted infections (STIs) remain a major global public health concern, with more than one million new cases acquired daily and increasing antimicrobial resistance compromising effective control strategies. Rapid, accurate and multiplex molecular diagnostics are therefore essential to support timely clinical management and public health surveillance. This study evaluated the analytical and clinical performance of the cartridge-based STANDARD™ M10 STI Panel (SD Biosensor, Republic of Korea), a fully automated, random-access real-time PCR assay capable of detecting eight STI-related pathogens within approximately 64 min. A total of 150 residual clinical specimens were retrospectively analysed, including vaginal, rectal, urethral and oropharyngeal swabs, as well as seminal fluids, and results were compared with those obtained using the Allplex™ STI Essential Assay (Seegene, Republic of Korea), which shares six common targets. The STANDARD™ M10 STI Panel demonstrated high diagnostic accuracy, with sensitivity ranging from 89.5% to 100%, specificity from 98.2% to 100% and overall accuracy between 98% and 100%. Agreement between the two assays was almost perfect, with Cohen’s κ values ranging from 0.91 to 1.00. Analytical sensitivity was further confirmed through verification of the limits of detection using quantified reference standards. Although validated for urine samples, the assay also showed robust performance on alternative clinical matrices, particularly vaginal swabs. Overall, these findings indicate that the STANDARD™ M10 STI Panel represents a reliable and practical tool for STI diagnosis, combining rapid turnaround time, minimal hands-on requirements and broad pathogen coverage in both centralized and near-patient testing settings. Full article

Background: Carbapenem-resistant Enterobacterales (CRE) are a global public health concern, with carbapenem-resistant Klebsiella pneumoniae (CR-Kp) recognised as the highest-priority pathogen. This study aimed to investigate the epidemiological features of CRE isolates throughout the COVID-19 pandemic in Buenos Aires, Argentina. Methods: A prospective study was conducted in two hospitals from 2019 to 2022, recovering all CRE from inpatients. Antimicrobial susceptibility was performed by automated and/or manual tests, according to CLSI. β-lactamases detection was performed using Multiplex PCR and MALDI-TOF MS. Kp typing was assessed by multiplex PCR and/or MLST based on WGS. Results: 22% (359/1594) were CRE, predominantly CR-Kp. Overall, high non-susceptibility (NS) rates were observed in both centres. NS remained largely stable in HA, except for a significant increase in colistin NS, whereas HB showed a rise in NS to multiple antimicrobials over time. A significant shift from multidrug-resistant to extensively drug-resistant and difficult-to-treat phenotypes was observed across the study periods. Out of 359 CRE, bla_KPC was confirmed in 141, bla_NDM in 170, and bla_KPC + bla_NDM in 20 isolates. Before the COVID-19 pandemic, KPC was the main carbapenemase in HB, while NDM was already the prevalent one in HA. In 2022, both enzymes showed similar prevalence. bla_KPC-2 and bla_NDM-5 were the prevalent alleles in K. pneumoniae. Before the COVID-19 pandemic, K. pneumoniae epidemiology varied by hospital, characterised by clonal diversity; however, in 2022, CG258-tonB79 drove the epidemiology in both hospitals. Conclusions: A more extensive resistance phenotype among CRE was evidenced throughout the COVID-19 pandemic, driven by carbapenemase-producing K. pneumoniae. NDM-5 and KPC-2 were the main carbapenemases identified. A temporal shift in carbapenemase prevalence was observed in each hospital, converging in similar frequencies of KPC and NDM by 2022 across both centres. This scenario was driven by the active dissemination of K. pneumoniae ST258. Full article

Hepatitis B virus (HBV) pregenomic RNA (pgRNA), transcribed directly from nuclear covalently closed circular DNA (cccDNA), is an essential component in viral replication. The synthesis and encapsidation of pgRNA depend significantly on the transcriptional activity of cccDNA, making serum pgRNA a recently recognized non-invasive biomarker for evaluating cccDNA activity. However, its clinical application is limited by factors including preanalytical variables, methodological inconsistencies in detection, and a lack of standardization in quantification. This review provides an overview of the biological origins of pgRNA and its critical role in the HBV replication cycle, highlighting the stability challenges encountered during the collection, processing, and storage of plasma/serum samples. Furthermore, it analyzes recent significant advancements in pgRNA detection technologies, encompassing modified reverse transcription quantitative polymerase chain reaction (RT-qPCR), nucleocapsid-captured methodologies, automated testing platforms, multiplex digital PCR, isothermal amplification, and clustered regularly interspaced short palindromic repeats-based assays. A comparison of these technologies revealed that discrepancies in pgRNA quantification arise primarily from variations in sample processing and measurement systems, rather than from inherent biological limitations. Therefore, establishing standardized sample handling procedures, harmonized detection methods, and unified measurement systems is imperative before pgRNA can be reliably applied to monitor treatment, guide cessation decisions, or evaluate cure in chronic hepatitis B. 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

Newborn screening (NBS) for spinal muscular atrophy (SMA) and severe combined immunodeficiency (SCID) faces challenges. Accurate and precise SMN1 and SMN2 copy number determination, confirmed by two orthogonal methods, are vital for SMA prognostication and treatment. Single SMN1 copy detection also enables the further feasibility to screen for compound heterozygotes. In SCID, low-level T-cell receptor excision circle (TREC) quantification by quantitative PCR is imprecise, necessitating replicates for reliable results. An assay with enhanced accuracy, precision, and high throughput is warranted for NBS SMA and SCID. False positive of SMN1 deletions due to allele dropout are also a potential pitfall in PCR-based methods. We evaluated a first-tier fully automated quadruplex droplet digital PCR (ddPCR) assay detecting SMN1, SMN2, TREC, and RPP30 using dried blood spots together with a second-tier Sanger sequencing to exclude SMN1 allele dropout. Five proficiency test samples and six patient samples with known SMN1 and SMN2 copy numbers confirmed by multiplex ligation-dependent probe amplification were used for accuracy evaluation with full concordance. The ddPCR assay showed high precision for SMN1 and SMN2 (<7% coefficient of variation (CV) for ≥0 copy) and TREC (14.6% CV at 37 copies/µL blood). Second-tier Sanger sequencing identified all SMA cases with homozygous deletions. Accuracy for TREC classification was concordant with 10 proficiency samples. The reference interval of TREC concentration was established for newborns ≥ 34 weeks (n = 1812) and the 2.5th percentile was 57 copies/µL blood. A two-tiered approach with fully automated quadruplex ddPCR and Sanger sequencing delivers accurate and precise quantitation for NBS SMA and SCID, enabling early treatment and counseling. Full article

Objective: This study aimed to validate the clinical utility of a salivary molecular platform (Oral Predict^®) for periodontal pathogen detection across preventive, therapeutic, and maintenance settings. Methods: A longitudinal randomized study was conducted involving 78 adults who provided saliva samples at baseline, one month, and three months after professional dental hygiene. Participants were randomized into two groups: control group (n = 39) and probiotic group with Oral Predict^® probiotic supplementation (n = 39). Crude saliva was processed directly without nucleic acid extraction and analyzed by multiplex real-time PCR using either the compact Real-time PCR system or standard thermocyclers. Results: At baseline, Fusobacterium nucleatum was the most prevalent pathogen (84.6%), followed by Tannerella forsythia (53.8%) and Porphyromonas gingivalis (46.2%). The Total Pathogen Burden Score (TPBS) showed progressive increases with age, smoking, and poor oral hygiene, and was significantly higher in participants with gingival bleeding. Among individual pathogens, no significant associations were observed with periodontitis staging or grading. Professional hygiene induced mean reductions of 1–2 logs across all pathogens, with TPBS decreasing from 8.7 ± 3.2 to 4.1 ± 2.8 (p < 0.001). At three months, 69.2% of the control group experienced bacterial rebound, whereas 85% of probiotic users sustained or improved bacterial reductions. Conclusions: Salivary molecular testing provides a robust, non-invasive approach for periodontal pathogen detection, treatment monitoring, and long-term maintenance assessment. The flexibility of the Oral Predict^® platform across point-of-care and laboratory settings, combined with automated interpretation, supports integration into preventive protocols and personalized periodontal care. These findings demonstrate the potential of saliva-based molecular diagnostics to shift periodontal management from reactive to predictive and precision-based strategies. Full article

Background/Objectives: We have previously reported the engineering of a point-of-care (POC) system that fully automates the procedures for nucleic acid extraction and multiplexed real-time RT-PCR, with a major advantage of high-level multiplexing. In this study, we applied and validated the system in a respiratory tract infection setting. Methods: An automatic nested real-time RT-PCR assay was developed (POCm). It was a 40-plex assay that simultaneously detected 39 epidemiologically important respiratory pathogens in 1.5 h in the POC system. The analytical and clinical performance was evaluated. Results: The analytical sensitivities of the POCm assay were comparable to those of its single-plex counterparts performed manually on a bench-top. The minimum detectable concentrations ranged from 53 copies/mL to 5.3 × 10³ copies/mL for all pathogen targets except hCoV-NL63 (5.3 × 10⁴ copies/mL). The quantitative performance was demonstrated by the linear correlations between Ct values and input concentrations for all pathogen targets, with 24 of them demonstrating coefficients of correlation (r) greater than 0.9. The POCm assay was subsequently evaluated in 283 clinical samples. A high level of agreement (98.2–100%) was achieved for pathogen detection results between POCm and standard diagnostic methods. The POCm result was also fully concordant with the result of another commercial POC multiplex platform. For positive clinical samples, pairwise Ct values measured by POCm closely correlated with those of the bench-top reference method (r = 0.70). The feasibility of mutation genotyping of the viral subtype was further demonstrated. Conclusions: This study demonstrated the practicality of POCm for routine testing in clinical laboratories. Further clinical trials are being conducted to evaluate the clinical performance of the system. Full article

Three targeted therapies are currently available for spinal muscular atrophy (SMA), which have dramatically changed the natural history of this severe and potentially fatal disease. More than 95% of SMA cases have a homozygous deletion of exon 7 of the SMN1 gene. Disease expression mainly depends on the copy number of SMN2, a hypomorphic copy of SMN1. Many countries in the world have implemented newborn screening (NBS) programs for early identification and treatment of children with SMA. We herein present the first two-year results of the SMA NBS program in Campania, a region with one of the highest birth rates in Italy. Genomic DNA was extracted from dried blood spots (DBS) and peripheral blood. For DBS, the SMN1 gene copy number was evaluated by quantitative polymerase chain reaction (qPCR) targeting SMN1 exon 7 and a reference gene (RPP30). In positive newborns and their parents, SMN1/SMN2 copies were evaluated by multiplex ligation probe amplification (MLPA). We analyzed 77,945 newborns and identified 11 positive children. Six patients had 2 copies of SMN2, but only one showed severe SMA-related signs at birth. Eligible newborns were treated with gene therapy within 20 days of birth. Notably, qPCR failed to amplify the reference RPP30 gene in 10/77,945 DBS. Despite this limitation, we observed that about 1/40 DBS had ΔCt values consistent with the presence of one SMN1 copy. The semi-automated procedure used for SMA NBS showed excellent performance in detecting the presence of homozygous deletion of SMN1 exon 7, with the exception of a few cases with the absence of amplification of the reference gene. By solving this limitation, the screening procedure has the potential to detect heterozygous carriers of the SMN1 deletion and, consequently, identify families at procreative risk of SMA. Full article

Background/Objectives: Due to the significant overlap in symptoms between COVID-19 and other respiratory infections, a multiplex PCR-based platform was developed to simultaneously detect 22 respiratory pathogens. Target sequences were retrieved from the GenBank database and aligned using Clustal Omega 2.1 to identify conserved regions prioritized for primer design. Primers were designed using Primer Express^® 3.0.1 and evaluated in Primer Explorer to ensure specificity and minimize secondary structures. A multiplex strategy organized primers into three groups, each labeled with distinct fluorophores (FAM, VIC, or NED), allowing for detection by conventional PCR or capillary electrophoresis (CE). Methods: After reverse transcription for RNA targets, amplification was performed in a single-tube reaction. A total of 340 clinical samples—nasopharyngeal and saliva swabs—were collected from patients, during the COVID-19 pandemic period. The automated analysis of electropherograms enabled precise pathogen identification. Results: Of the samples analyzed, 57.1% tested negative for all pathogens. SARS-CoV-2 was the most frequently detected pathogen (29%), followed by enterovirus (6.5%). Positive results were detected in both nasopharyngeal and saliva swabs, with SARS-CoV-2 predominating in saliva samples. Conclusions: This single-tube multiplex PCR-CE assay represents a cost-effective and robust approach for comprehensive respiratory pathogen detection. It enables rapid and simultaneous diagnosis, facilitating targeted treatment strategies and improved patient outcomes. Full article

Background/Objectives: Concurrent outbreaks of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza A and B viruses (IAV/IBV), and respiratory syncytial virus (RSV) necessitate rapid and precise differential laboratory diagnostic methods. This study aimed to evaluate the multiplex molecular diagnostic performance of the geneLEAD VIII system (Precision System Science Co., Ltd., Matsudo, Japan), a fully automated sample-to-result precision instrument, in conjunction with the VIASURE SARS-CoV-2, Flu & RSV Real Time PCR Detection Kit (CerTest Biotec, S.L., Zaragoza, Spain). Methods: The specific detection capabilities of SARS-CoV-2, IAV/IBV, and RSV genes were evaluated using virus-spiked saliva and nasal swab samples. Using saliva samples, the viral titer detection limits of geneLEAD/VIASURE and manual referent singleplex RT-qPCR assays were compared. The performance of geneLEAD/VIASURE in analyzing single- and multiple-infection models was scrutinized. The concordance between the geneLEAD/VIASURE and the manual assays was assessed. Results: The geneLEAD/VIASURE successfully detected all the virus genes in the saliva and nasal swab samples despite some differences in the Ct values. The viral titer detection limits in the saliva samples for SARS-CoV-2, IAV, IBV, and RSV using geneLEAD/VIASURE were 10⁰, ≤10⁻², 10⁰, and 10² TCID₅₀/mL, respectively, compared to ≤10⁻¹, ≤10⁰, ≤10⁰, and ≤10⁴ TCID₅₀/mL, respectively, in the manual assays. geneLEAD/VIASURE yielded similar Ct values in the single- and multiple-infection models, with some exceptions noted in the triple-infection models when low titers of RSV were spiked with high titers of the other viruses. The concordance between geneLEAD/VIASURE and the manual assays was high, with Pearson’s R² values of 0.90, 0.85, 0.92, and 0.95 for SARS-CoV-2, IAV, IBV, and RSV, respectively. Conclusions: geneLEAD/VIASURE is a reliable diagnostic tool for detecting SARS-CoV-2, IAV/IBV, and RSV in single- and multiple-infection scenarios. Full article

Staphylococcus aureus is a leading cause of foodborne intoxication globally, driven by its heat-stable enterotoxins (SEs), which pose significant public health risks. This review critically evaluates modern and traditional methodologies for detecting S. aureus and its enterotoxins in food matrices, emphasizing their principles, applications, and limitations. The review includes a dedicated section on sample preparation and pretreatment methods for diverse food substrates, addressing a critical gap in practical applications. Immunological techniques, including ELISA and lateral flow assays, offer rapid on-site screening but face matrix interference and variable sensitivity challenges. Molecular methods, such as PCR and isothermal amplification, provide high specificity and speed for bacterial and toxin gene detection but cannot confirm functional toxin production. Sequencing-based approaches (e.g., WGS and MLST) deliver unparalleled genetic resolution for outbreak tracing but require advanced infrastructure. Emerging biosensor technologies leverage nanomaterials and biorecognition elements for ultra-sensitive real-time detection, although scalability and matrix effects remain hurdles. Mass spectrometry (MALDI-TOF MS) ensures rapid species identification but depends on pre-isolated colonies. Traditional microbiological methods, while foundational, lack the precision and speed of molecular alternatives. The review underscores the necessity of context-driven method selection, balancing speed, sensitivity, and resource availability. Innovations in multiplexing, automation, AI-based methods, and integration of complementary techniques are highlighted as pivotal for advancing food safety surveillance. Standardized validation protocols and improved reporting of performance metrics are urgently needed to enhance cross-method comparability and reliability in outbreak settings. Full article

Background: Microscopy is the conventional method for the identification of gastrointestinal parasitic pathogens in fecal specimens; however, it presents numerous challenges, including high technical expertise burden, multiple staining procedures, and prolonged turnaround time. Molecular methods provide higher throughput and potentially higher sensitivity and specificity. Methods: We validated a commercial, automated DNA extraction platform and multiplex parasitic real-time PCR panel (Seegene Allplex^TM GI-Parasite Assay) detecting six protozoal pathogens: Blastocystis hominis (Bh), Cryptosporidium spp., Cyclospora cayetanensis (Cc), Dientamoeba fragilis (Df), Entamoeba histolytica (Eh), and Giardia lamblia (Gl) in unpreserved fecal specimens submitted for diagnostic parasitology. Microscopy was the reference standard for all organisms, with stool ELISA as an additional reference assay for Eh. Results: Among 461 unpreserved fecal specimens, sensitivity, specificity, positive predictive and negative predictive values of the enteric multiplex for fresh specimens were as follows: 93%, 98.3%, 85.1%, 99.3% for Bh; 100% for all measures in Cryptosporidium and Cc; 100%, 99.3%, 88.5%, 100% for Df; 33.3%, 100%, 100%, 99.6% for Eh; and 100%, 98.9%, 68.8%, 100% for Gl, respectively. With the addition of 17 frozen specimens, the sensitivity for Eh increased to 75%. On a per-batch basis, the molecular platform reduced pre-analytical and analytical testing turnaround time by 7 h. Conclusions: The enteric multiplex platform provides a useful diagnostic tool for clinically relevant enteric protozoa, including Cryptosporidium spp., Cyclospora cayetanensis, Dientamoeba fragilis, and Giardia lamblia. Further evaluation of the assay is required for Entamoeba histolytica prior to clinical use; however, given the widespread availability of confirmatory serology and stool antigen testing for E. histolytica, such performance limitations are of lesser concern. Full article

Viral meningitis poses a significant clinical challenge due to its rapid onset and potential progression to life-threatening encephalitis. Early detection of treatable viral pathogens such as Herpes simplex virus (HSV), Cytomegalovirus (CMV), and Varicella-zoster virus (VZV) is essential for initiating appropriate therapies. However, multiplex PCRs for the rapid and simultaneous detection of these pathogens are scarce due to the complex PCR design and the elaborate validation process using cerebrospinal fluid samples. In this study, we established and validated a novel multiplex PCR assay for detecting HSV, CMV, and VZV in cerebrospinal fluid samples and implemented the assay on a fully automated platform. Full article

Background/Objectives: DNA methylation is a key epigenetic mark involved in regulating gene expression. Aberrant DNA methylation contributes to various human diseases, including cancer, autoimmune disorders, atherosclerosis, and cardiovascular diseases. While whole-genome bisulfite sequencing and methylated DNA immunoprecipitation (MeDIP) are standard techniques for studying DNA methylation, they are typically limited to a few samples per run, making them expensive and low-throughput. Therefore, an automation-friendly method is needed to increase throughput and reduce costs without compromising data quality. Methods and Results: We developed a novel method called Multiplexed Methylated DNA Immunoprecipitation Sequencing (Mx-MeDIP-Seq), which can be used to analyze many DNA samples in parallel, requiring only small amounts of input DNA. In this method, 10 different DNA samples were fragmented, purified, barcoded, and pooled prior to immunoprecipitation. In a head-to-head comparison, we observed a 99% correlation between MeDIP-Seq performed individually or combined as Mx-MeDIP-Seq. Moreover, multiplexed MeDIP led to more than 95% normalized percent recovery and a 25-fold enrichment ratio by qPCR, like the enrichment of the conventional method. This technique was successfully performed with as little as 25 ng of DNA, equivalent to 3400 to 6200 cells. Up to 10 different samples were processed simultaneously in a single run. Overall, the Mx-MeDIP-Seq method is cost-effective with faster processing to analyze DNA methylome, making this technique more suitable for high-throughput DNA methylome analysis. Conclusions: Mx-MeDIP-Seq is a cost-effective and efficient method for high-throughput DNA methylation analysis, offering faster processing and reduced sample requirements. This technique makes DNA methylome analysis more accessible for large-scale studies. Full article