Chikungunya virus (CHIKV) represents a significant global health threat due to its recent resurgence. This protocol highlights the use of capillary-modified visual loop-mediated isothermal amplification (LAMP) technology for the rapid detection of CHIKV. We employed Vero cells for virus propagation and utilized the MolPure Viral DNA/RNA Kit for nucleic acid extraction, successfully amplifying extracted viral RNA at 63°C for 25 min. The presence of CHIKV was indicated by a distinct yellowish-green color change, demonstrating the effectiveness and simplicity of this protocol. Our protocol underscores the potential of LAMP technology as a rapid diagnostic tool for CHIKV, enabling timely public health interventions.
Methods in molecular biology (Clifton, N.J.)Pooja Bhardwaj, Gaurav Raj Dwivedi, Hari Shanker Joshi, Rajeev Singh
Chikungunya virus (CHIKV) is transmitted through the bite of Aedes mosquitoes, specifically A. aegypti and A. albopictus. CHIKV belongs to the alphavirus with a positive-sense ssRNA genome of 11-12 kb size. The virus has been reported from various geographical regions across the globe. Chikungunya fever is an acute febrile illness, which, if left untreated, may develop into chronic arthralgia that may persist for several months or acute encephalitis syndrome. Therefore, early diagnosis of CHIKV is crucial to initiate prompt supportive treatment. Laboratory diagnosis of CHIKV typically relies on serological tests such as IgM antigen capture ELISA and molecular methods including RT-PCR or qRT-PCR. However, both these methods are not viable in peripheral settings. This chapter highlights recent advancements in molecular detection techniques for CHIKV, specifically isothermal detection methods that eliminate the requirement for complex instruments. The detection is facilitated by RT-RPA and CRISPR/Cas12a endonuclease. The assay offers advantages over existing methods such as rapid and early detection, and eliminates cross-over contamination, ultra-sensitivity, high specificity, and ease of result interpretation.
Science advancesDavid J Roach, Bhrus Pun Sangruji, Shriya Bhat, Selama Tesfamariam, Ishay Ben-Zion, Miriam Bern, Josephine Bagnall, Noam Shoresh, Lisa Milien, Roby P Bhattacha…
Antimicrobial resistance is a major global health threat, with disproportionate impact in regions with limited diagnostic infrastructure. To address this challenge, we developed BADLOCK (Bacterial and AMR Detection by SHERLOCK), a rapid, low-cost molecular diagnostic platform for direct detection of bacterial pathogens and resistance genes from clinical samples. BADLOCK operates as a one-pot CRISPR-Cas13a reaction capable of detecting nine bacterial species and four major resistance genes directly from positive blood culture. It requires only a heat block and a basic fluorometer for operation. We validated BADLOCK on 194 sequentially collected clinical blood culture specimens growing Gram-negative rods, supplemented with 69 mock samples generated from banked isolates enriched for targeted resistance genes. In all, we conducted 2224 individual reactions, achieving 97.6% accuracy (2171 of 2224) at the reaction level. At the assay level, 89.5% (274 of 306) showed perfect or partial concordance with gold-standard species and resistance gene detection, including 255 assays with perfect concordance and 19 with partial concordance (correct detection of at least one pathogen). This included an evaluation of BADLOCK as a potential culture-free diagnostic for urinary tract infections (UTIs), achieving 98.0% reaction-level accuracy. At the assay level, 95.3% (41 of 43) were partially or perfectly concordant with gold-standard detection of both species and resistance genes. To our knowledge, this represents the first demonstration of the CRISPR-Cas13a diagnostic platform on clinical bloodstream infections to date and supports BADLOCK's potential as a practical and scalable solution for rapid pathogen and resistance gene detection in resource-constrained settings.
Indian pediatricsAbhishek Purohit, Pallab Chatterjee, Hema Mittal, Madhu S Pujar, Sanjiv Singh Rawat, Pranita Pradhan, Joseph L Mathew
CONTEXT: Determining the microbial etiology in children with empyema thoracis can guide optimal treatment of individual children, and rational antimicrobial use in institutions. Culture methods and molecular diagnostic technologies are available, but there is no evidence-based guideline addressing the optimal diagnostic technology. OBJECTIVE: This systematic review was undertaken to compare molecular diagnostic methods with culture methods for determining microbial etiology in children with empyema. EVIDENCE ACQUISITION: Literature search was undertaken in seven databases and four clinical trial registries, to identify diagnostic test accuracy (DTA) studies or diagnostic randomized controlled trials, comparing molecular methods with culture, for establishing the microbiological etiology of empyema. The outcomes were diagnostic accuracy parameters. Risk-of-bias and certainty of evidence were evaluated using the GRADE approach. RESULTS: Nine DTA studies were included, however data from eight could be pooled. The pooled sensitivity of polymerase chain reaction (PCR) in pleural fluid specimens was 0.98 (95% CrI 0.91-0.99), and specificity 0.49 (95% CrI 0.19-0.75). The likelihood ratio of a positive result (LR+) was 1.90 (95% CrI 1.20-3.88), whereas LR- was 0.05 (95% CrI 0.01-0.23). Positive predictive value ranged from 0.11 to 1.0, and negative predictive value from 0.90 to 1.0. The diagnostic odds ratio was 38.23 (95% CrI 5.66-231.81). The overall risk of bias and overall applicability concerns were low in 3/8 studies and unclear in 5/8. The certainty of evidence was very low. CONCLUSION: Pleural fluid PCR has high sensitivity compared to culture, but moderate specificity. The optimal choice needs considerations beyond the comparative diagnostic accuracy.
Emerging infectious diseasesCarlos Abelardo Dos Santos, Jialu Li, Pauline M van Diemen, Andrew McMahon, Benjamin C Mollett, Andrew M Ramsay, Meshach M Maina, Joe James, Helen E Everett, J…
The Orthomyxoviridae family includes influenza D virus (IDV), an emerging pathogen primarily affecting cattle and swine; there is evidence of cross-species transmission and potential zoonotic risk. Although active human infections have yet to be confirmed, high seroprevalence in cattle-exposed populations highlights the need for continued surveillance. We developed and validated a rapid, field-deployable reverse transcription loop-mediated isothermal amplification assay for IDV detection; specificity was 99.2% and sensitivity ranged from 95.6% (cycle quantification <30) to 81.8% (cycle quantification <40). This method offers a cost-effective, accessible alternative to quantitative reverse transcription PCR, enabling improved monitoring of IDV and reinforcing preparedness for emerging influenza threats.
Antimicrobial resistance (AMR) constrains effective treatment and carries implications for infection control, surveillance, and public health. The World Health Organization (WHO) priority bacterial pathogen framework has intensified the need for diagnostic innovation by redefining research priorities around organisms combining high disease burden with complex resistance profiles. Molecular diagnostics have accordingly moved beyond culture-based workflows, integrating rapid pathogen identification, resistance-marker detection, genomic surveillance, and clinical decision support. The present study conducted a bibliometric mapping of the literature on WHO priority pathogens. Rather than addressing resistance at a general level or a single pathogen or technology, it integrates priority pathogens, molecular platforms, and resistance markers within a single framework, tracing their joint thematic and temporal evolution along an explicit pathogen-platform-marker axis. Scopus-indexed articles and reviews (2000-2025) were retrieved, yielding 1746 publications after screening adapted from the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. Analyses used Bibliometrix/Biblioshiny, R, and VOSviewer. The literature expanded markedly after 2018, led by China and the United States. Methicillin-resistant Staphylococcus aureus (MRSA), Mycobacterium tuberculosis, Enterococcus faecium, and the Enterobacterales-carbapenemase axis constituted the principal thematic cores, whereas conventional polymerase chain reaction (PCR)/nucleic acid amplification testing (NAAT) and whole-genome sequencing were the dominant platforms. Overall, the field has evolved from pathogen detection into an AMR-centered translational domain encompassing resistance prediction, genomic epidemiology, surveillance, and clinical decision support. Diagnostic development, stewardship, and surveillance depend on hybrid workflows coupling rapid marker-targeted assays with genome-based characterization, delivering actionable resistance within clinically meaningful timeframes, and extending coverage to underrepresented pathogens and platforms.
Hematology/oncology clinics of North AmericaJulia A Elvin
Over the past three decades, the landscape of clinically available molecular tests has evolved due to advancements in basic science cancer research and the subsequent utilization of this knowledge to develop DNA, RNA, and protein-based molecular assays for oncology that can be employed for routine clinical use in diagnostics laboratories. Molecular testing of tumors is revealing gaps in previous histopathologic classification systems and opportunities for new, personalized treatment paradigms. Awareness of validated molecular assay options and their general advantages and limitations is crucial for oncology care providers to ensure the optimal test(s) are selected for each patient's circumstances.
BACKGROUND: Molecular testing (MT) aids in the management of indeterminate thyroid nodules; however, regional differences remain unexamined. This meta-analysis compares MT's clinical impact between Western and Asian cohorts. This study hypothesized that regional disparities in malignancy prevalence, molecular platforms, and practice patterns would yield divergent clinical applications of MT. METHODS: A systematic review up to April 2025 identified studies reporting the surgical management and histopathological outcomes of indeterminate nodules in Asian and Western cohorts, stratified by use of MT versus no MT. Pooled proportions were calculated via random-effects models. RESULTS: Of 127 included studies (100 Western; 27 Asian), MT was performed in 96 Western and 21 Asian studies. Western studies predominantly used commercial panels, whereas Asian studies favored smaller, mutation-specific assays. Without MT, resection rates were 66.8% (95% CI, 55.1%-76.7%) in Western studies versus 53.9% (95% CI, 43.5%-64.0%) in Asian studies (p = .11). With MT, rates fell to 42.5% versus 37.4% (p = .275), respectively. Risk of malignancy (ROM) was significantly higher in Asian cohorts, both without MT (48.0% vs. 29.5%; p = .005) and with MT (62.7% vs. 37.1%; p < .001), which persisted after exclusion of follicular thyroid neoplasm with papillary-like nuclear features. In Western studies, second-generation platforms achieved lower resection rates (36.1% vs. 48.3%; p = .001) but higher ROM (42.8% vs. 34.4%; p = .005) than first-generation tests. CONCLUSIONS: MT reduces surgical interventions across both regions by serving divergent roles. In the West, MT functions as a rule-out tool to avoid surgery via comprehensive panels. In Asia, where baseline malignancy is higher and practice is relatively conservative, MT provides confirmatory risk stratification via focused mutation panels. Regionally tailored guidelines are warranted.
BACKGROUND: Hereditary ataxias are clinically and genetically heterogeneous, which complicates clinical management, genetic counseling, and scientific research. Efficient molecular diagnosis is essential but requires molecular epidemiological considerations due to the variable genetic landscapes across regions and populations. In China, despite the established predominance of spinocerebellar ataxia type 3 (SCA3), comprehensive data on the genetic landscape of hereditary ataxias are limited and increasingly outdated, leaving insufficient evidence to support an epidemiology-informed molecular diagnostic strategy and its clinical implementation as a diagnostic algorithm. METHODS: We retrospectively established a large, real-world, comprehensive cohort of hereditary ataxias (the HARMONY cohort) recruited from two medical centers in the Yangtze River Delta region of China (2008-2024) for genetic profiling. Molecular diagnoses were categorized into four classifications ('Definitive', 'Probable', 'Unclear', 'Undiagnosed') by integrating previously identified causative variants with results from systematic genetic testing in etiologically undetermined probands, and the associated variants were mapped onto genetic landscapes. RESULTS: A total of 2096 probands were included in the HARMONY cohort, achieving a molecular diagnostic yield of 84.9%, with 1715 (81.8%) 'Definitive' and 65 (3.1%) 'Probable' positive diagnoses, alongside 87 (4.2%) 'Unclear' and 229 (10.9%) 'Undiagnosed' cases. While the genetic landscape was dominated by variants underlying SCA3 (1249; 59.6%), with SCA2 (150; 7.2%), SCA1 (139; 6.6%), SCA6 (51; 2.4%), putative SCA36 (36; 1.7%), and SCA12 (35; 1.7%) constituting the major subtypes, the genetic profiling revealed a long tail of rare genotypes. Onset age distributions were characterized for genotypes with ≥ 5 probands. No probands were found to carry expansions in ZFHX3, BEAN1, DAB1, THAP11, or FXN. CONCLUSIONS: Genetic profiling of the HARMONY cohort revealed a skewed genetic landscape dominated by a few common repeat-expansion subtypes, alongside a markedly heterogeneous long tail of rare etiologies. The holistic profile supports a stepwise molecular diagnostic strategy, under which we propose a YRD (Yangtze River Delta)-oriented candidate diagnostic algorithm. This study provides an evidence base for early molecular diagnosis, prioritization of therapeutic development, and adaptive design of diagnostic tools for hereditary ataxias.
BACKGROUND: Chromosome 19p13.3 microdeletion syndrome is a rare and clinically heterogeneous genomic disorder. It is typically characterized by a wide spectrum of manifestations, including global developmental delay, various congenital anomalies, and distinct craniofacial dysmorphisms. Current literature suggests that phenotypic severity and the specific symptoms are highly dependent on the precise size and gene content of the deleted region. METHODS AND RESULTS: Here we report the case of an 8-month-old female infant referred for evaluation of multisystemic complications. Clinical examination revealed a complex phenotype characterized by congenital heart defects, limb anomalies, macrocephaly, and recurrent pulmonary infections. Oligo-Array CGH revealed a 1.8 Mb deletion encompassing 55 genes. Although gastrointestinal symptoms have rarely been reported in this condition, the patient presented with gastrointestinal complications including vomiting and diarrhea. CONCLUSION: This case expands the clinical spectrum of 19p13.3 microdeletion syndrome and underscores the importance of genomic analysis in understanding the underlying disease mechanisms.
SLAS technologyMadhusudan B Kulkarni, Vineela, Naresh Mandal, Yashwanth N, Panwala Fenil Chetankumar, Sergio O Martínez-Chapa, Ricardo Izquierdo
Nucleic acid detection has gained significant attention in healthcare because of its crucial role in diagnosing infectious and non-infectious diseases. Conventional molecular diagnostics are predominantly performed in centralized laboratories using sophisticated instrumentation and automated analyzers, resulting in increased analysis time, cost, and limited accessibility in decentralized settings. Consequently, there is an increasing demand for rapid, cost-effective, portable, and user-friendly diagnostic platforms capable of providing reliable results at the point of care. Target nucleic acid detection commonly relies on amplification strategies, including thermocyclic amplification such as polymerase chain reaction (PCR) and various isothermal amplification techniques. Although these methods provide high sensitivity and specificity, challenges associated with instrumentation, thermal control, sample preparation, assay integration, and accessibility remain. Microfluidic integration provides a promising approach for addressing these limitations by enabling miniaturized sample handling, reduced reagent consumption, rapid amplification, and integrated detection within compact analytical platforms. This review critically examines recent advances in microfluidic nucleic acid biosensors, including PCR and isothermal amplification platforms, emerging amplification and detection technologies, device integration, and point-of-care applications. Further, emphasis on comparative analytical performance, technological limitations, and the remaining challenges associated with translating microfluidic nucleic acid detection systems from laboratory demonstrations to practical point-of-care diagnostics applications.
Functional nucleic acids can connect molecular recognition with chemical signal generation, but their diagnostic value depends on the performance of the complete sample-to-answer pathway. This critical narrative review examines representative studies published through 31 July 2026, with emphasis on recognition, amplification, material interfaces, sample preparation, readout and clinical interpretation under realistic conditions. Hybridization and ligation probes, aptamers, DNAzymes, DNA nanostructures and CRISPR-associated systems are compared by specificity, kinetics, leakage and matrix compatibility. Rolling circle amplification, hybridization chain reaction, catalytic hairpin assembly and enzymatic isothermal amplification are evaluated as reaction networks whose products must remain accessible to the selected interface. Functional materials are classified by their actual analytical role, including transduction, signal amplification, capture/enrichment, spatial organization and reagent storage. Evidence is distinguished between mechanistic studies, spiked matrices, clinical specimens, manufactured-format reproducibility and demonstrated clinical utility. We further integrate sample-to-answer workflow, assay time, complexity, regulatory considerations and clinically relevant decision thresholds. Across the literature, reliable performance depends on selective recognition before high-gain reactions, compatibility between amplification products and interfaces, explicit controls for inhibition and leakage, and validation across independent lots and representative clinical populations. These principles define a path from analytical proof of concept to reproducible and clinically interpretable diagnostic testing.
World journal of urologyMeghana Reddy, Christina Sze, Bonnie C Prokesch, Philippe Zimmern
INTRODUCTION: Recent advances have led to a rise in metagenomic and molecular-based testing for more rapid diagnosis of urinary tract infections (UTI). We conducted a contemporary systematic review to understand how these molecular tests affect clinical outcomes. METHODS: A systematic review was conducted for articles in adults from January 2023 to October 2025 as an update to previous scoping reviews. Cochrane and PRISMA standards were utilized with the following databases: PubMed, OVID, and Embase (PROSPERO number: CRD420251070022). Data was extracted from the selected full-text papers including the type of study (microbiology versus clinical), patient demographics, key findings, and funding source. Excluded studies were abstracts, non-English manuscripts, and those involving only men or children. RESULTS: The search resulted in 12 full-text articles. Among those, 7 were published in clinical-based journals and 5 were in Microbiology. Two clinical studies were randomized controlled trials (RCTs) which included a total of 773 patients, mostly female and above the age of 65. Compared to a standard urine culture, molecular based testing, specifically with polymerase chain reaction (PCR) testing yielded superior sensitivity despite lack of specificity and guided antibiotic therapy to reduce UTI baseline symptomatology in the short term (i.e. 28 days) in both RCT analyses. Of note, the RCT analyses and several of the prospective studies (5/10) were funded by the companies that developed the molecular tests. CONCLUSIONS: Based on the increase in publications over the last three years, new industry-funded RCTs, and the limited clinical outcome data reported, this systematic review indicates the urgent need for prospective and multicentric studies to better understand the role of these molecular tests in UTI management.
NeurogeneticsFlavia Privitera, Stefano Pagano, Giulia Nutile, Camilla Meossi, Roberta Battini, Mariapaola Schifino, Filippo Maria Santorelli
Deletions of the short arm of chromosome 2 are rare, and phenotypical differences depend on the cytobands involved. Here, we present a new patient carrying a de novo heterozygous 2p13-p11.2 deletion, detected by array CGH and further investigated by optical genome mapping. We compare his clinical findings with few other similar cases described in the literature, and with those associated with 2p12-p11.2 deletions. We conclude that the two conditions are clinically overlapping, and propose genes not previously implicated, such as TET3 and CCT7, may aggravate their main clinical features in the context of a contiguous multigenic deletion syndrome.
Journal of visualized experiments : JoVEXudong Liu, Liang Bai, Qianlin Wang, Giovanni Scimeca, Qian Liang, Xiaoxin Li, Lei Wang, Longxiang Su
Lower respiratory tract infections (LRTIs) are among the leading causes of clinical deterioration and death in critically ill patients. In the intensive care unit (ICU), timely identification of the causative pathogen is essential for appropriate antimicrobial therapy; however, conventional culture methods take 3 to 5 days and have suboptimal positivity rates, particularly for fungi. Molecular approaches such as PCR shorten turnaround time but still depend on manual nucleic acid extraction and thermocycling, limiting their suitability for bedside use. This protocol describes a point-of-care workflow built around a fully integrated, disc-shaped microfluidic chip (CD chip) that couples magnetic bead-based nucleic acid purification with loop-mediated isothermal amplification (LAMP). After a simplified manual liquefaction and lysis step, the processed specimen is loaded onto the chip together with two prepackaged reagent vials. The instrument then autonomously performs extraction, amplification at 65 °C, and real-time fluorescence readout, returning qualitative results for 15 targets, namely 11 bacteria, 3 fungi, and 1 atypical pathogen, within 45 min of instrument run time (approximately 1 h for the complete sample-to-answer workflow, including the manual preprocessing step). We present detection data from 10 ICU specimens, including sputum, bronchial aspirates, and bronchoalveolar lavage fluid, encompassing both single-pathogen and polymicrobial co-infections involving up to 7 organisms on a single chip. The closed cartridge design minimizes the risk of aerosol contamination, and the single loading step requires no specialized training, positioning this system as a practical bedside diagnostic tool for ICU teams.
Journal of otolaryngology - head & neck surgery = Le Journal d'oto-rhino-laryngologie et de chirurgie cervico-facialeErika Lee, Rana Chakrabarti, Norbert Viallet, Alok Pathak, Suhail Sayed, Paul Kerr, Ciaran Lane
OBJECTIVE: To determine the cost-effectiveness of molecular testing for indeterminate (Bethesda III/IV) thyroid nodules in a real-world, single-payer healthcare system. DESIGN: This retrospective population-based cohort study analyzed fine-needle aspiration biopsy (FNAB) and lobectomy data for thyroid nodules in Manitoba, Canada, during 2023. Microcosting was performed to capture surgical, surveillance, molecular testing, and complication costs in 2023 Canadian dollars. Six molecular tests (Afirma, ThyroSeqV3, ThyroidPrint, ThyroSPEC, ThyGeNEXT/ThyraMIR, and mir-THYpe) were evaluated using site-specific malignancy prevalence (21%) and published test statistics. SETTING: The study was performed within a publicly funded healthcare system. PARTICIPANTS: Participants were 18 years and above having undergone FNAB in Manitoba.Intervention or Exposures:Three scenarios were identified Current Care, Molecular Testing, and Reference Standard (diagnostic lobectomy) pathways. The total cost per year were compared among scenarios. MAIN OUTCOME MEASURES: Cost-utility analyses compared Current Care, Molecular Testing, and Reference Standard pathways. Quality-adjusted life-years (QALYs) and incremental cost-effectiveness ratios (ICERs) were calculated. A $50 000/QALY willingness-to-pay (WTP) threshold was used to interpret ICERs. RESULTS: Among 1486 FNABs performed, 413 were indeterminate nodules, and 149 patients underwent diagnostic lobectomy. The cost of lobectomy was $7542.69 per patient. All molecular testing strategies improved net QALYs and demonstrated ICERs ranging from $9791.03 to $58 749.94 per QALY compared to Current Care. All tests apart from Afirma were cost-effective at the WTP threshold relative to Current Care. Compared to the Reference Standard, all molecular testing strategies were dominant. CONCLUSIONS: For the investigated Manitoba scenario, molecular testing for indeterminate thyroid nodules can be a cost-effective strategy, possibly reducing unnecessary surgeries while preserving patient outcomes. RELEVANCE: This study contributes to the body of evidence suggesting molecular testing is cost-effective and has potential to influence political decisions in health care spending.
Pathogens (Basel, Switzerland)Fabiola Mancini, Enza Bruna Bonazza, Antonino Bella, Antonietta Girolamo, Massimo Mentasti, Maria Luisa Ricci, Maria Scaturro
The laboratory diagnosis of Legionnaires' disease (LD) remains a significant challenge due to the limitations of current methods. Most LD cases are diagnosed through the detection of Legionella urine antigen using commercial assays that are usually specific just for Legionella pneumophila (Lp) serogroup 1. Isolating Lp through culture is rarely done and difficult to achieve, especially due to two important issues: the low sensitivity of the method and the fact that it is difficult to obtain respiratory secretions from a significant proportion of patients. Real-time PCR (qPCR) has significantly improved LD diagnosis, indicating that molecular methods based on Legionella DNA detection can be highly effective. This study aimed to adapt a qPCR assay to a digital PCR (dPCR) assay, taking advantage of the more sensitive and more sophisticated dPCR technology. In particular, the study focused on 42 respiratory specimens, including bronchoalveolar lavage, tracheal aspirates, and sputum samples, with a particular focus on culture-negative specimens presumed to contain low concentrations of Legionella DNA, which were considered particularly suitable for assessing the potential of dPCR to improve the diagnosis of Legionnaires' disease. The results of culture, qPCR, and dPCR of respiratory samples were compared with each other and with data from urinary antigen tests (UATs) available for the corresponding patients. Overall, dPCR detected Legionella DNA in additional respiratory specimens compared with qPCR, corresponding to a 19% increase in the number of qPCR-negative specimens yielding a positive dPCR result. dPCR also detected Legionella DNA in specimens from patients with negative urinary antigen results. These findings suggest that dPCR may have potential as a complementary molecular approach for improving Legionella DNA detection in respiratory specimens, particularly in culture-negative samples. However, the clinical significance of dPCR-positive/qPCR-negative results could not be independently confirmed in this study and requires further investigation.
PulmonologyMalaisamy Muniyandi, Sahil Abdul Salam, Venkateshprabhu Janagaraj, Priya Rajendran, Silla Varghese Thomas, Kavitha Rajsekar, Karikalan Nagarajan
BACKGROUND: The molecular platforms such as Truenat and GeneXpert have enhanced tuberculosis (TB) diagnostic capacity in India. However, these technologies still face challenges related to cost, infrastructure requirements and maintenance. To address these gaps, several newer molecular diagnostic tools are emerging, including UniAMP. However, the cost implications and economic feasibility remain limited in Indian context. We conducted cost comparison of UniAMP with Truenat for TB detection in India. METHODS: A decision tree model was developed from a health system perspective to compare UniAMP with Truenat for TB detection. Sensitivity and specificity data were collected from published literature and validation reports. Diagnostic outcomes such as true positives and negatives were estimated for the cohort of 1,000 presumptive TB patients. Costs data were collected from ICMR-NIRT laboratory and average cost per test and per case detection was estimated. The comparison was done between UniAMP and Truenat. RESULTS: The estimated per test cost was ₹483 (US$5.54) for UniAMP and ₹834 (US$9.57) for Truenat. Case detection in a cohort of 1,000 presumptive TB, true positive was 265 and 296; true negatives 647 and 651 in UniAMP and Truenat respectively. UniAMP identified fewer true positive TB cases than Truenat (265 vs 296), resulting in 31 (10.7%) reduction in diagnostic yield. CONCLUSIONS: UniAMP may support decentralised TB testing in resource limited settings because of its lower cost and minimal infrastructure requirements. UniAMP may therefore be more suitable as a supplementary diagnostic option rather than a replacement for established molecular tests such as Truenat.
Frontiers in cellular and infection microbiologyZhenghan Luo, Minzhi Xu, Letian Zhang, Yifang Han, Anqi Tan, Taiwu Wang, Fuqiang Ye, Chunhui Wang, Jinhai Zhang
BACKGROUND/OBJECTIVES: Novel influenza A viruses (NIAV) infection is a zoonotic acute respiratory disease. The H5N1 and H7N9 subtypes cause high mortality, posing major public health threats. This study aimed to establish rapid, field-deployable subtype-specific nucleic acid detection assays for H5 and H7 by integrating reverse-transcription enzymatic recombinase amplification (RT-ERA) with CRISPR/Cas12a. Two separate reactions are required per sample. METHODS: Conserved hemagglutinin (H) sequences were retrieved from the NCBI database, and primers, probes, and crRNAs were designed using Primer Premier 5 and Primer-BLAST. The sensitivity of RT-ERA alone was compared with that of the ERA-CRISPR/Cas12a using serially diluted virus strain nucleic acid. The specificity was verified against other respiratory pathogens with similar clinical manifestations. RESULTS: The optimal primers were F1R2 for H5 and F2R3 for H7. The limit of detection (LOD) of RT-ERA alone was 2.63×10³ copies/µL for H5 and 1.86×10³ copies/µL for H7; the ERA-CRISPR/Cas12a assay improved the LOD to 2.63×10¹ copies/µL for H5 and 1.86 copies/µL for H7, representing 100-fold and 1000-fold enhancements in sensitivity, respectively. Specificity testing showed that the assay exclusively detected H5N1 and H7N9 with no cross-reactivity to other tested pathogens. CONCLUSION: The ERA-CRISPR/Cas12a method exhibits enhanced sensitivity for H5 and H7 subtypes detection, along with high specificity, rapidity, and minimal equipment requirements. It offers a promising screening tool for NIAV infections.
International journal of molecular sciencesYoungmin Han, Hye Jin Yoo
Hypertension (HTN) is often asymptomatic and difficult to detect using blood pressure (BP) measurements unless BP is substantially elevated. Given its association with metabolic alterations and complications, this study aimed to establish a metabolite risk score (MRS) as a molecular tool to complement BP-based diagnosis. Plasma samples and clinical data from healthy individuals and HTN patients were obtained through the Korea Biobank Network, and non-targeted metabolomics was performed. Eight HTN-associated key metabolites were selected by least absolute shrinkage and selection operator (LASSO) regression. An MRS was calculated as their weighted sum in the discovery set and subsequently validated in the replication set. The MRS showed strong discriminative performance for HTN status in the replication set [area under the curve (AUC) = 0.926, 95% confidence interval (CI): 0.876-0.976] and remained significantly associated with prevalent HTN after adjustment for age and BMI [odds ratio (OR) = 1.747, 95% CI: 1.317-2.318, p < 0.001]. At the MRS cut-off, classification accuracy in the replication set was approximately 84%, with 83.7% sensitivity and 84% specificity. The MRS also showed weak-to-moderate positive correlations with systolic BP in both the discovery set (r = 0.320, p = 0.001) and the replication set (r = 0.335, p < 0.001); however, these correlations were no longer statistically significant after adjustment for age and BMI. These findings support further evaluation of the MRS as a complementary molecular approach for HTN discrimination. Integration with other omics platforms may facilitate the development of more comprehensive molecular approaches for HTN. Further validation in larger prospective longitudinal cohorts is required before its potential clinical application.
World journal of microbiology & biotechnologyJunxue Qiu, Yanping Qi, Xuelong Chen
Listeria monocytogenes remains a foodborne pathogen of major global public health concern, associated with high mortality, particularly in developing countries where food safety surveillance infrastructure is limited. The hly gene, which encodes the pore-forming toxin listeriolysin O, is a highly conserved virulence determinant across all major serotypes. In this study, we developed a closed-tube isothermal multiple self-matching-initiated amplification (IMSA) assay targeting the hly gene for the specific and visual detection of L. monocytogenes. Optimized to run at 63 °C for 45 min, the assay employs SYBR Green I preloaded in the tube cap, enabling direct visual readout under UV light without the post-amplification tube opening-a feature critical for resource-limited laboratories. The assay detected all six L. monocytogenes strains tested (serotypes 1/2a, 1/2c, 4b, and 4e) without cross-reactivity against 11 non-target foodborne pathogens. In phosphate-buffered saline (PBS), the limit of detection (LOD) was 1 × 102 CFU/mL, matching quantitative real-time PCR (qPCR) and outperforming loop-mediated isothermal amplification (LAMP) tenfold. This analytical sensitivity was determined in PBS; complex food matrices may elevate the practical detection limit owing to the presence of inhibitory substances. When applied to 84 enriched retail food samples from Anhui Province, China-spanning dairy, meat, and seafood-the assay identified 17 positive samples (20.24%) and showed complete concordance with the culture-based reference method. This closed-tube IMSA assay provides a rapid, specific, and equipment-minimal screening tool suitable for food safety monitoring in basic laboratories, particularly in developing regions lacking access to qPCR instrumentation.
Frontiers in cellular and infection microbiologyDarwin Paredes-Núñez, Silvia Salgado, Martha Sánchez, Katherine Natalia Chávez-Toledo, Johanna Parrales, Andrés Tinizaray, José Julián Zúñiga-Velarde, Solón Al…
Mpox virus (MPXV) is an enveloped double-stranded DNA virus belonging to the genus Orthopoxvirus within the family Poxviridae. It causes a zoonotic disease transmitted to humans through direct contact with infected animals or humans, as well as through exposure to contaminated materials. The aim of this study was to conduct a retrospective, comparative diagnostic evaluation of three commercial real-time PCR (qPCR) kits for the diagnosis of MPXV on an enriched, artificially balanced sample set that included 50% MPXV-positive samples. World Health Organization (WHO) reference molecular protocol was used as the reference comparator. A total of 300 clinical specimens, 150 of them MPXV-positive (265 skin lesion/vesicle swabs and 35 serum samples), were used in this analysis. The Bioperfectus kit correctly identified 294/300 samples, showing a sensitivity of 0.96 (95% CI: 0.93-0.99), a specificity of 1.00 (0.98-1.00), and a Cohen's Kappa coefficient of 0.96 (95% CI: 0.92-0.99), with an overall agreement of 0.98 (0.96-0.99). The VIASURE kit correctly detected 293/300 specimens, yielding a sensitivity of 0.95 (0.92-0.99), specificity of 1.00 (0.98-1.00), Cohen's Kappa coefficient of 0.95 (95% CI: 0.92-0.99 and an overall agreement of 0.98 (0.95-0.99). The Singuway kit showed the highest concordance with the reference method, correctly classifying 295/300 samples and presenting a sensitivity of 0.97 (0.94-1.00), specificity of 1.00 (0.98-1.00), Cohen's Kappa coefficient of 0.97 (95% CI: 0.94-1.00), with an overall agreement of 0.98 (0.97-1.00). This study demonstrates a good sensitivity and specificity, and strong overall agreement among the three commercial kits compared to the WHO protocol. Nevertheless, further analysis using unbiased clinical sample sets that reflect the true prevalence of MPXV-positive samples is needed for a real-world clinical performance evaluation.
BiosensorsShaoshuang Sun, Xueping Zhou, Xiuling Yang
Mulberry mosaic dwarf-associated virus (MMDaV), a member of the genus Mulcrilevirus in the family Geminiviridae, poses a serious threat to mulberry cultivation and growth in China. Early and accurate diagnosis is a prerequisite for the effective prevention and control of MMDaV-related disease. In this study, a loop-mediated isothermal amplification (LAMP)-based detection method was established for the specific identification of MMDaV. Three pairs of specific primers were designed targeting the nucleotide sequences of the MMDaV V2 gene. The optimized LAMP reaction was performed at a constant temperature of 65 °C for 60 min, and the established method exhibited high specificity with no cross-reactivity observed against other tested geminiviruses. The LAMP method achieved a detection limit of 200 pg of target MMDaV DNA, which was tenfold more sensitive than conventional PCR. Furthermore, the amplification results could be directly visualized via distinct color change. Collectively, the developed LAMP method enables efficient, sensitive, and specific isothermal detection of MMDaV and holds great promise for rapid diagnosis of mulberry viral disease.
Scientific reportsNatalia Dos Santos, Francisco de Menezes Cavalcante Sassi, Rodrigo Zeni Dos Santos, Caio Augusto Gomes Goes, Renata Luiza Rosa de Moraes, Fabio Porto-Foresti, …
B chromosomes are supernumerary elements that evolve from standard A chromosomes and are primarily composed of repetitive DNAs, yet their origin, diversification, and molecular composition remain poorly understood in most vertebrates. We investigated two allopatric populations of Cyphocharax modestus (Curimatidae) combining classical cytogenetics, comparative genomic hybridization (CGH), and comparative satellitomics to characterize the repetitive DNA landscape of its B chromosomes. While both populations exhibited a conserved karyotype of 2n=54 biarmed chromosomes, five individuals from the Batalha River (BR) carried supernumerary chromosomes, comprising two distinct variants: a C-positive B1 and an C-negative B2. Comparative satellitome analysis between 3B-carrying and B-lacking individuals identified 116 satellite DNAs (CmoSatDNAs), with the 3B library showing higher abundances of specific sequences. Fluorescence in situ hybridization (FISH) revealed that both B variants share two centromeric satellites (CmoSat01-192 and CmoSat02-108) with the A complement, while CmoSat58-47 was exclusively to B2. Minimum spanning tree analysis of CmoSat58-47 revealed B-exclusive haplotypes alongside haplotypes shared with B-lacking individuals, suggesting a recent origin for these chromosomes. CGH experiments further confirm the sequence sharing between the A and B chromosomes, supporting an intraspecific origin, and revealing substantial genomic differentiation among B variants.
Journal of visualized experiments : JoVEMarcos Francisco Hernández-Robles, Iván Natividad-Bonifacio, Ana Karen Álvarez-Contreras, José Carlos Parada-Fabián, Carlos Ramón Vázquez-Quiñones, Carlos Vázq…
The Vibrio genus has more than 100 species, of which 12 are considered pathogenic for humans; species most frequently isolated in food are V. cholerae, V. parahaemolyticus, V. vulnificus, and V. alginolyticus. The main problem of identifying them through conventional microbiological methods is the high costs and long-term diagnosis. Loop-mediated isothermal Amplification (LAMP) was used for the identification of V. cholerae, V. parahaemolyticus, V. vulnificus, and V. alginolyticus. Activated carbon with bentonite (ACB) was used to remove inhibitors, improving the sensitivity of the test; pH 6, 7, and 9 were tested, as well as different contact times between the sample and ACB. The LAMP sensitivity method was improved when activated carbon and bentonite were used to remove reaction inhibitors in samples of shrimp, fish, and oyster. LAMP sensitivity, negative predictive value, positive predictive value, and LAMP specificity were equal to or greater than 90% for all samples tested. The LAMP method could be an alternative to identify V. cholerae, V. parahaemolyticus, V. vulnificus, and V. alginolyticus in fishery products due to faster application, higher sensitivity, and lower costs than the standard method.
Analytical chemistryYanming Liu, Xiaotian Ma, Jianyu Hu, Gregory J Tyrrell, Petrus J van der Walt, Xing-Fang Li, X Chris Le
Isothermal amplification reactions have diverse applications in molecular diagnostics, point-of-care detection, and synthesis of nucleic acids. Isothermal amplification techniques eliminate the need for temperature cycling and the related equipment. However, a common issue of isothermal amplification is the formation of nonspecific amplification byproducts that cause false-positive detection. We report here the development and design principles of sequence-specific minor groove binder (MGB) and locked nucleic acid (LNA) probes, which significantly improve the detection specificity. We show an example of integrating loop-mediated isothermal amplification (LAMP) with the MGB and LNA probes and demonstrate an application of the integrated method to the specific detection of Streptococcus pyogenes (group A streptococcus [GAS]) and skin and wound specimens. The technique reduced background and eliminated false positives generated by nonspecific amplification byproducts. The method achieved a detection limit of 25 copies of the target speB DNA with a 30 min LAMP-probe reaction. Results from the analysis of 171 skin and wound specimens were consistent with clinical diagnostics of GAS infections, confirming 100% positive predictive value (PPV) and 100% negative predictive value (NPV) of the assay. The modular design of LAMP and the MGB and LNA detection probes can be readily adapted for the detection of other nucleic acid targets.
Tropical animal health and productionNikita Kaushik, Mansi Joshi, Rubina Kumari Baithalu, Rajani Kumar Paul, Dheer Singh, Suneel Kumar Onteru
Buffaloes do not exhibit overt estrus signs particularly during summer, leading to a significant economic loss to farmers. Previous studies have identified several candidate transcripts (HSP70, TIMP1, TLR4 and HSD17B1), abundant in buffalo saliva during estrus stage. However, there is no widely applicable technology for estrus detection targeting these transcripts. Therefore, the present study aimed to develop reverse transcription loop mediated isothermal amplification (RT-LAMP) assays for these candidate transcripts using buffalo saliva. Saliva samples were collected from 10 cyclic buffaloes and RT-LAMP assays were optimized for salivary RNA as well as direct saliva. Among the four candidate transcripts, HSP70 showed a statistically significant colour change (p-value = 0.0191) at the estrus stage compared to the diestrus stage. This abundance of HSP70 was also supported in large simulated population datasets (10,000 animals) generated using R. Further, the RT-LAMP assays were tested using direct saliva without RNA isolation, and the colour change in the samples during estrus suggested the feasibility of estrus identification using direct saliva, overcoming the tedious step of RNA isolation. The detection of HSP70 using either direct saliva or salivary RNA indicated its potential as a marker for estrus identification. Similarly, TLR4 appeared to be another potential biomarker for RT-LAMP reaction using direct saliva, but it needs further validation in both RNA and direct saliva samples. Overall, the proof-of-concept on RT-LAMP assays optimized for salivary transcripts in the present study would be useful for estrus identification in tropical production systems following further validation on a larger sample size.
Signal transduction and targeted therapyDing-Kun Ji, Haozhi Lei, Jia Liu, Fangfang Xia, Xiaobo Gao, Yi-Qing Zhou, Ruiying Li, Yongqi Han, Ruowen Wang, Weihong Tan
Nucleic acid aptamers, often referred to as "chemical antibodies," are versatile, specific, and easily modifiable functional nucleic acids. There is a growing focus on new methods for the selection and target validation of aptamers, with the aim of expanding their biomedical applications in molecular diagnostics and therapeutics, which is currently a research hotspot. This review is composed of eight sections. In the first section, we briefly introduce aptamers and review their development in molecular diagnostics and therapeutics. The "Advantages of aptamers in molecular diagnosis and therapeutics" section summarizes and discusses the advantages of aptamers in these fields. The "New methods for screening aptamers" section presents and discusses nucleic acid aptamer screening methods, including both classical and novel approaches. In the "New methods for target validation" section, we explore new methods for target validation, covering aptamer structure validation, target recognition validation, and aptamer-target interaction validation. The "New methods for molecular diagnostics" section summarizes and discusses recent applications of aptamers in molecular diagnostics, particularly focusing on new mechanisms and detection strategies as well as their applications in various diseases. The "New methods for molecular therapeutics" section summarizes and discusses recent applications of aptamers in molecular therapeutics, emphasizing new mechanisms and aptamer-based therapy strategies, along with their therapeutic applications in different diseases. The "Challenges and future perspectives of nucleic acid aptamers" section addresses the challenges and future perspectives of aptamers in disease diagnosis and treatment. Finally, the "Conclusion" section shares our views on the future directions of aptamers in clinical disease molecular diagnostics and therapeutics.
Women's health (London, England)Sandra Olivia Frans, Ariane Utomo, Linda Rae Bennett
BackgroundIndonesia has the highest rates of cervical cancer mortality in Southeast Asia, mainly due to poor access to and low uptake of cervical screening. Indonesian midwives are primarily responsible for conducting cervical screening, yet there is very limited research on their relevant knowledge, training and capacity to provide screening.ObjectiveThis study explored midwives' experiences of cervical screening training and their knowledge related to CC, cervical screening using Visual Inspection with Acetic Acid (VIA), and Human Papillomavirus (HPV) DNA testing in East Nusa Tenggara, Indonesia.DesignA qualitative study design was employed, based on primary data collected through fieldwork using semi-structured interviews and supplemented by a knowledge assessment questionnaire.MethodsTwenty-four midwives across two districts, one semi-urban and one rural, participated. Qualitative data were analysed using a codebook thematic analysis approach, and questionnaire data were descriptively analysed.ResultsMidwives' mean knowledge score was 75%, with midwives trained in cervical screening scoring higher than untrained midwives. Our findings highlighted inequitable access to training, with midwives in the more urban district having more access to training and higher knowledge. Midwives emphasised that adequate training and continuous practice of the VIA were crucial to building confidence in service delivery. Key knowledge gaps included poor understanding of the relationships between HPV infection, pre-cancer lesions, cervical cancer and its prevention. Midwives' poor knowledge of cervical cancer aetiology and asymptomatic pre-cancer lesions resulted in only recruited women who came with associated symptoms.ConclusionsGeographical inequality in human resource investment in remote areas limits women's access to quality cervical screening and impedes progress towards cervical cancer elimination. Supporting midwives through thorough and up-to-date training, including HPV DNA testing, is essential to increase screening access and uptake, and for the transition to HPV DNA testing.
Journal of primary care & community healthChayanis Kositamongkol, Euarat Mepramoon, Sukrit Kanchanasurakit, Pattarawalai Talungchit, Pattama Chaopotong, Kirati Kengkla, Thanet Chaisathaphol, Surasak Sa…
ObjectivesAlthough cervical cancer screening is available to all Thai women, incidence and mortality remain high because uptake remains low. The national health benefits package now covers self-collected human papillomavirus (HPV) DNA testing, which may reduce barriers. We explored the perceived feasibility and acceptability of self-collected HPV DNA testing within Thailand's national cervical cancer screening policy.MethodsWe conducted individual, in-depth interviews with 40 participants to explore their perspectives. Participants were healthcare providers involved in cervical cancer screening (n = 20) and women aged 25-65 years targeted by the program (n = 20). Interviews were conducted at a university hospital in Bangkok, Thailand, and guided by tailored open-ended question lists for each group.ResultsMost participants perceived self-collected HPV DNA testing as acceptable and potentially feasible for reducing barriers and increasing screening uptake. Reported concerns included inaccurate specimen collection, transport difficulties, and risk of injury. Participants emphasized that public outreach and education for both women and providers are essential to policy success. They also stressed that women at risk, or with gynecologic symptoms, must be examined by a clinician. Providers further called for a consensus clinical-practice protocol for cervical cancer screening.ConclusionsSelf-collected HPV DNA testing was perceived as acceptable and was supported by both providers and target women. Raising awareness of the importance of screening is critical to maximizing uptake. However, self-collected HPV DNA testing cannot replace clinical assessment of symptomatic women. Rather, it should be offered as an additional option to increase participation among non-attenders.
Frontiers in cellular and infection microbiologyYonghui Gao, Jihong Chen
BACKGROUND: Mosquito-borne viral diseases pose an important public health threat due to their frequent outbreaks, similar early clinical manifestations, and potential for rapid transmission. Conventional diagnostic methods, such as virus isolation, serological assays and RT-qPCR, are reliable but may be limited by long turnaround time, cross-reactivity, laboratory equipment requirements and trained operators. Recombinase polymerase amplification (RPA)-based methods have emerged as promising tools for rapid detection of mosquito-borne viruses, but their overall diagnostic accuracy still requires comprehensive evaluation. This meta-analysis aimed to systematically evaluate the diagnostic performance of RPA-based assays for mosquito-borne virus detection. METHODS: A systematic search of PubMed, Embase, Web of Science, Scopus and MEDLINE was conducted using search terms related to mosquito-borne viruses, RPA technology and diagnostic accuracy. Studies reporting sensitivity and specificity or providing sufficient data to construct 2 × 2 contingency tables were included. The methodological quality of included studies was assessed using QUADAS-2. R software was used for statistical analysis. A Bayesian bivariate random-effects model was applied to calculate pooled sensitivity, specificity, positive likelihood ratio (PLR), negative likelihood ratio (NLR) and summary receiver operating characteristic (SROC) curve. RESULTS: Ten studies involving 13 data sets in total were included in this meta-analysis. The pooled sensitivity and specificity of RPA-based assays were 0.96 (95% CrI: 0.92-0.98) and 0.99 (95% CrI: 0.98-1.00), respectively. The pooled PLR was 146.25 (95% CrI: 35.79-597.57), and the pooled NLR was 0.05 (95% CrI: 0.02-0.10). The area under the SROC curve was 0.987 (95% CrI: 0.972-0.995), suggesting excellent overall diagnostic performance. Subgroup analyses showed that RPA-based assays maintained good diagnostic accuracy in both CRISPR-based and non-CRISPR groups, as well as in DENV and non-DENV subgroups. No significant publication bias was detected by Deeks' funnel plot asymmetry test. CONCLUSION: RPA-based assays showed good diagnostic accuracy for mosquito-borne virus detection, with high pooled sensitivity, specificity and AUC. These findings suggest that RPA-based methods may provide a useful technical choice for rapid case identification and public health response. Nevertheless, the limited number of included studies, methodological limitations and insufficient real-world evidence warrant cautious interpretation. More high-quality prospective studies are still needed to confirm their practical feasibility and application value in clinical and public health settings. SYSTEMATIC REVIEW REGISTRATION: https://www.crd.york.ac.uk/prospero/, identifier CRD420261380107.
BACKGROUND: External Quality Assessment Schemes (EQAS) are essential for ensuring the accuracy, reliability, and comparability of molecular diagnostic testing across laboratory networks. To strengthen dengue molecular surveillance and laboratory quality systems in India, a Pan-Dengue Molecular Serotyping EQAS was developed and implemented among Virus Research and Diagnostic Laboratories (VRDLs) in India. METHODS: A nationwide EQAS was conducted in three rounds between December 2024 and February 2026. Proficiency testing panels consisting of five coded lyophilized serum samples (500 µL each) containing dengue-positive and dengue-negative specimens were prepared, characterized, validated, and distributed to participating laboratories. Homogeneity and stability assessments were performed prior to distribution. Participating laboratories tested the panels using their routine molecular diagnostic workflows and reported dengue detection and serotype identification results through the National Institute of Epidemiology (NIE) PT Panel Portal. Laboratory performance was evaluated based on concordance with assigned reference results. RESULTS: A total of 34, 40, and 56 laboratories enrolled in Round 1, Round 1 Phase-II, and Round 2, respectively. The corresponding response rates were 91.2%, 95.0%, and 94.6%. Complete concordance was achieved by all reporting laboratories in Round 1 (31/31) and Round 1 Phase-II (38/38). In Round 2, 50/53 laboratories (94.3%) achieved 100% concordance, while two and one laboratory achieved 80% and 60% concordance, respectively. The overall pass rate across all rounds was 97.5%. CONCLUSION: The Pan-Dengue Molecular Serotyping EQAS highlighted a high level of proficiency among participating laboratories and effectively strengthened molecular diagnostic quality assurance within the national laboratory network. Regular implementation of such programmes will support reliable dengue surveillance, continuous quality improvement, and enhanced preparedness for dengue outbreaks in India.
Clinics in laboratory medicineJaylou M Velez Torres, Youley Tjendra, Darcy A Kerr
Risk stratification is essential in the preoperative evaluation and management of thyroid nodules, most of which are benign. Advances in DNA and RNA sequencing have shed light on the molecular drivers of thyroid cancer. Molecular testing of cytologically indeterminate nodules has helped refine risk stratification, triage patients for surgery, and determine the extent of surgery. Molecular platforms with high negative predictive values can help identify nodules that may be spared surgery and can be managed conservatively. Here we discuss the importance of integrating cytomorphologic, molecular, and histologic features to help avoid errors and improve patient management.
Veterinary medicine and scienceSeyed Ali Ghorashi, Jiongrui Huang, Peter Scott, Sameer D Pant
BACKGROUND: Rapid and reliable diagnostic methods suitable for field use are needed for the detection of Erysipelothrix rhusiopathiae in poultry. OBJECTIVES: This study aims to develop and evaluate a colourimetric loop-mediated isothermal amplification (LAMP) assay for the rapid detection of E. rhusiopathiae in conjunction with a rapid DNA extraction procedure suitable for field conditions. METHODS: Specific primers targeting a conserved region of a putative polypeptide gene involved in the synthesis of capsular polysaccharides were used for LAMP and PCR. The specificity of the developed assays was evaluated using 13 unrelated bacterial strains. The efficacy of the assays was assessed using positive controls and 52 clinical samples collected from poultry farms experiencing erysipelas. RESULTS: Both LAMP and PCR showed high sensitivity and specificity when compared with bacterial culture. LAMP and PCR achieved 100% sensitivity and specificity when combined with the rapid DNA extraction method. Using a commercial DNA extraction kit, LAMP maintained 100% sensitivity and specificity, whereas PCR showed 100% sensitivity and 97.8% specificity. The high correlation observed between bacterial culture and the LAMP assay demonstrated the effectiveness and reliability of the rapid DNA extraction method. CONCLUSIONS: The developed colourimetric LAMP assay provides a rapid, sensitive, and practical diagnostic tool for the detection of E. rhusiopathiae in poultry. The assay requires minimal instrumentation, enables straightforward visual interpretation of results, and is suitable for point-of-care testing and resource-limited settings.
United European gastroenterology journalGiannoula S Tansarli, Ferric C Fang
Multiplex polymerase chain reaction (PCR) panels are increasingly used for the evaluation of acute diarrhea, providing rapid and sensitive detection of a broad range of enteric pathogens. However, their widespread adoption has created important interpretive challenges for clinicians. Positive results may reflect colonization, prolonged shedding, or organisms of uncertain pathogenicity rather than active infection, and current panels do not provide antimicrobial susceptibility or quantitative information. Incomplete pathogen coverage and false-positive detections may further complicate result interpretation, particularly in patients with mild or self-limited disease. Inappropriate test ordering or overinterpretation of results may lead to unnecessary antimicrobial therapy, delayed evaluation of non-infectious causes of diarrhea, and increased healthcare utilization. This review summarizes the key limitations of multiplex GI PCR panels and provides a practical framework for integrating molecular results with clinical presentation, disease severity, and patient risk factors to support informed management decisions in everyday clinical practice.
Journal of infection in developing countriesNianhong Lu, Jin Guo, Caihong Liu, Jiangyuan Wang, Danhua Wang, Xue Li, Ye Yuan
OBJECTIVE: To evaluate the diagnostic performance and clinical utility of targeted third-generation sequencing (tTGS) for pathogen detection in community-acquired pneumonia (CAP), compared with targeted next-generation sequencing (tNGS) and conventional culture. METHODOLOGY: We conducted a real-world, retrospective study including 356 CAP patients who were tested in parallel with culture, tNGS, and tTGS. Diagnostic sensitivity, pathogen spectrum, mixed-infection detection, and antimicrobial resistance (AMR) gene profiling were compared across methods. Clinical relevance was assessed by analyzing treatment adjustments informed by sequencing results. RESULTS: tTGS demonstrated the highest overall sensitivity (99.16%), surpassing tNGS (96.35%, p < 0.05) and culture (50.27%, p < 0.01). Across all samples, tTGS identified 135 microbial species - substantially more than tNGS (84 species) and culture (27 species) - including fastidious, rare, and slow-growing pathogens. tTGS also showed an improved ability to detect mixed infections (86.24% vs. 87.64% with tNGS; culture detected none). AMR gene detection was significantly higher with tTGS than with tNGS (58.99% vs. 48.88%, p = 0.008), and long-read sequencing enabled the identification of composite resistance patterns that tNGS missed. Among patients without underlying diseases, sequencing-guided therapeutic adjustment occurred in 67.87% of cases, with nearly 80% of AMR-positive patients demonstrating clinical improvement following regimen optimization. CONCLUSIONS: tTGS provides markedly enhanced pathogen detection, greater sensitivity, and more comprehensive AMR profiling than both tNGS and culture. Its strong performance in identifying mixed infections and actionable determinants of resistance supports its incorporation into clinical diagnostic workflows for CAP. Further large-scale, prospective studies are warranted to validate its clinical impact and optimize its integration into routine practice.
Journal of infection in developing countriesYan Shen, Si-Ming Lu, Hong-Liang Liu, Jia-Long Wang, Yang Li, Li-Guo Liang
INTRODUCTION: The aim of this study was to develop a rapid isothermal amplification assay for Vibrio cholerae. METHODOLOGY: Specific primers were designed following the guidelines of the National Center for Biotechnology Information (NCBI) database and based on the highly conserved sequence of the Vibrio cholerae virulence factor gene ctxA. A detection method was then developed using recombinase polymerase amplification (RPA). Sensitivity, specificity, clinical sample collection, and processing were calculated. RESULTS: A rapid isothermal amplification system was developed, showing a detection capability of one copy per microliter. Specificity analysis against a panel of control strains yielded no false-positive results under the tested conditions. The presence of V. cholerae was detected in 9 fecal samples collected from 20 patients who were hospitalized with suspected cholera infection. CONCLUSIONS: This method can be an effective aid in the detection of bacteria in clinical samples.
Frontiers in public healthAnna R Bird, Jacqueline C Linnes, Laud Anthony Basing, Elizabeth A H Hall
Diagnostics are not only tools for clinical decision making, but also drivers of the healthcare supply chain. They quantify and monitor the prevalence of disease across global populations. At high levels, this population health data is used to inform the purchasing and distribution of health care goods. There is overwhelming evidence that access to diagnostics is limited in low-and middle-income countries. This is true for both in-vitro molecular diagnostics and diagnostic imaging, but the scope of this work is limited to the former. While the factors contributing to limited access are numerous, interconnected, and complex, we propose a method to alleviate some restraints within the supply chain: distributed and local manufacturing of key biological reagents, or active diagnostics ingredients (ADIs). In addition to shifting the geographical boundaries of manufacturing and moving goods closer to areas of deployment, this could reduce global power imbalances by redistributing ownership of diagnostics goods, reagents, and intellectual property.
Journal of virological methodsMin Young Lee, Vu Minh Phan, Hiep Van Nguyen, Woo In Lee, Tae Seok Seo
Rapid and accurate identification of respiratory viruses is essential for effective clinical management and infection control. However, conventional RT-qPCR methods are time-consuming and require complex workflows. To address these limitations, we developed a novel Switching RT-qPCR assay designed for multiplex detection of respiratory viruses with enhanced efficiency and sensitivity. This study aimed to evaluate the analytical and clinical performance of the switching RT-qPCR assay in the detection of seven major respiratory viruses: Influenza A and B, respiratory syncytial virus (RSV) A and B, adenovirus (AdV), human metapneumovirus (hMPV), and SARS-CoV-2 (Severe acute respiratory syndrome coronavirus 2). Nasopharyngeal swab specimens (n = 808) were collected from patients with respiratory symptoms. RNA was extracted and tested using both a Switching RT-qPCR assay and commercial multiplex RT-qPCR method (Allplex Respiratory Panels and Allplex SARS-CoV-2 Assay). The concordance, sensitivity, specificity, and analytical performances of the two methods were compared. The Switching RT-qPCR assay demonstrated a clinical sensitivity of 93.6% and a specificity of 100%, with an overall concordance rate exceeding 96%, compared with the conventional assay. Analytical sensitivity was comparable or superior across most viral targets, particularly RSV A, AdV, and hMPV. The duplex assay format enabled the efficient simultaneous detection of two targets per reaction, reducing the overall testing time while maintaining analytical accuracy. This Switching RT-qPCR assay offers a reliable, rapid, and efficient alternative for respiratory virus detection. Its simplified duplex format and high concordance with standard molecular diagnostics indicate its potential utility in routine clinical laboratories.
International journal of molecular sciencesSaranthum Phurijaruyangkun, Pongbun Tangjitrungrot, Kantima Choosang, Adun Bunchaleamchai, Naiyana Wattanakul, Bajaree Jantrapanukorn, Rungnapa Veeramano, Suph…
Carbapenem-resistant Acinetobacter baumannii (CRAB) is a major cause of hospital-acquired infections. Conventional phenotyping for carbapenem resistance requires more than 24 h, which delays clinical decisions and increases the risk of outbreaks. This study aims to develop a lateral flow dipstick (LFD) assay that detects loop-mediated isothermal amplification (LAMP) products targeting the blaOXA-23-like gene, providing results within 1-1.5 h. Primer specificity was evaluated using seven Gram-positive and nine Gram-negative species commonly present in clinical samples, as well as carbapenem-susceptible A. baumannii and CRAB isolates harboring other resistance genes. The assay detected the blaOXA-23-like gene at concentrations as low as 1.57 pg/µL, demonstrating ~10-fold greater sensitivity than conventional PCR. The gene was present in 224/268 CRAB samples (83.6%) and absent in 100 carbapenem-susceptible A. baumannii samples. Results were fully concordant with PCR, yielding 100% sensitivity and specificity. The method requires no expensive or complex instrumentation. Future studies should validate the assay across multiple hospitals and clinical specimens to confirm its generalizability and clinical utility. Multiplex LAMP with dual LFD readouts could also enable simultaneous detection of additional carbapenem resistance genes, supporting rapid CRAB surveillance and outbreak control.