Analytical methods : advancing methods and applicationsYan Qing, Hai-Bo Wang, Qin Huang, Qing Liu, Lei Xie, Yi-Mei Wu, Xin Lu, Qun-Ying Zhao, Ya-Jing Dong, Man Li, Ru-Qin Yu, Hao Tang
Rapid diagnosis of viral diseases in pepper (Capsicum annuum L.) is crucial for preventing pathogen spread, reducing yield losses, and supporting the green and sustainable development of the pepper industry. Here, we present an integrated, low-cost centrifugal microfluidic chip that simultaneously detects nucleic acids from six pepper viruses. All reagents are pre-stored on-chip as dried powders and are used to perform real-time fluorescence reverse transcription loop-mediated isothermal amplification (RT-LAMP). The developed assay showed strong agreement with conventional diagnostic methods of polymerase chain reaction (PCR). This platform offers a practical approach for rapid nucleic acid-based diagnostics and epidemiological monitoring of pepper viral diseases.
Analytical methods : advancing methods and applicationsLéo Baldenweck, Noémie Berg, Mila Djisalov, Vitaly Efremov, Zorica Novakovic, Sandrine Auger, Jasmina Vidic
Rapid identification of bacteria and their virulence factors is essential for global public health. Isothermal amplification has become a cornerstone of point-of-care diagnostics, enabling genetic testing to be faster, simpler, and more accessible than culturing or polymerase chain reaction (PCR). This review examines recent advances in some of the most commonly used isothermal amplification methods for bacterial detection: SDA, LAMP, HDA, RPA, RCA, and NASBA. The integration of isothermal amplification with the CRISPR/Cas system or microfluidic devices is also highlighted as an advanced gene detection technology. We present various readout methods used to detect gene amplification products or processes, including colorimetric, fluorescent, electrochemical, and quartz microbalance techniques. These integrated approaches can detect very small amounts of bacterial DNA, in under an hour, providing rapid, sensitive, versatile, and portable tools for health control.
PloS oneIsabelle Robène, Mikel Arrieta-Salgado, Samuel Rozsasi, Diane Mostert, Véronique Maillot-Lebon, Yolande Chilin-Charles, Jos Jansen van Vuuren, Gloria Valentine…
Bacterial diseases of banana are a growing global threat, causing yield losses and increased management costs. Major diseases include Moko, banana blood disease (BBD), and banana Xanthomonas wilt (BXW), caused by Ralstonia solanacearum, Ralstonia syzygii subsp. celebesensis, and Xanthomonas vasicola pv. musacearum, respectively. Effective surveillance requires point-of-care diagnostics such as loop-mediated isothermal amplification (LAMP) for on-site use. We aimed to develop three LAMP assays to specifically detect the bacteria responsible for Moko, BBD and BXW, directly from banana tissues, using a simplified DNA extraction protocol. The BBD - and BXW-LAMP assays demonstrated 100% specificity, yielding negative results for a broad range of non-target bacteria, including closely related species as well as pathogenic and endophytic strains associated with banana, and positive results for all the tested target strains. For Moko disease, a duplex-LAMP assay was developed to detect all strains from the four globally most relevant sequevars: IIB-3, IIB-4, IIA-6, and IIA-24. The duplex-LAMP successfully detected all target strains, except one that was shown to be non-pathogenic to Cavendish bananas. All non-target strains tested negative, with the exception of a delayed signal for one strain belonging to Ralstonia thomasi, not associated with banana environment (hospital strain). These results were supported by an extensive in silico analysis conducted on 9,668 Burkholderiaceae and 7,483 Xanthomonadaceae genomes. Detection limits ranged from 0.1 pg/µl to 1 pg/µl DNA, and from 104 to 105 CFU/ml on banana tissues spiked with calibrated bacterial suspensions, depending on the assay. The LAMP assays prove highly effective for detecting target pathogens in both artificially inoculated banana plants and field samples, offering a promising tool for improving disease management strategies.
Urinary tract infections (UTIs) are among the most prevalent bacterial infections worldwide, accounting for a major clinical impact due to high recurrence rates, microbial diversity, biofilm formation, polymicrobial contribution and the rapid emergence of antimicrobial resistance (AMR). Traditional culture-based diagnostic approaches are constrained by high turnaround times and insufficient resolution of virulence and resistance factors, frequently result in empirical antibiotic therapy. Recent breakthroughs in molecular biology and biotechnology have fuelled the advancement of innovative diagnostic approaches for the quick, sensitive and pathogen-specific detection of uro-pathogens. Rapid pathogen identification and antibiotic susceptibility testing are critically needed for the effective targeted antibiotic therapy. This paper initially examines promising technologies employing machine learning models to provide rapid diagnostic outcomes, specifically for urinary tract infections. This paper focuses on promising molecular diagnostic technologies such as nucleic acid amplification, biosensor-based platforms, microfluidic lab-on-chip systems and omics-based approaches, along with their amalgamation with Artificial intelligence (AI) and smart diagnostics. Improved diagnostic precision, recommendations for targeted antimicrobial therapy and support for antimicrobial resistance surveillance and management are among the translational applications of these developments that are highlighted. Challenges related to medical ethics, execution and regulations are also covered. The combined efforts of next-generation and AI-assisted diagnostic tools offer a revolutionary paradigm for accurate and efficient antibiotic resistance control to treat UTIs.
Over the past several decades, diagnostic microbiology has progressed from traditional culture methods to include modern, culture-independent molecular and metagenomic approaches for diagnosing infectious diseases and guiding antimicrobial therapy. Since the beginning of the twenty-first century, clinical diagnostic microbiology has made considerable strides in optimizing pathogen identification. This progress has been driven by the introduction of optimized sampling methods, advanced diagnostic kits, and new technologies like mass spectrometry for bacterial identification, real-time genomics, and adaptable culture systems. However, the costs of advanced molecular methods are very high, and they require massive instrumentation to reach a clinical diagnosis. Conventional cultures remain cost-effective and can be performed with minimal resource requirements compared to advanced laboratory equipment. However, the most significant challenge with conventional methods is the reporting time of results (several days). Since the newer molecular and genomic methods do not meet all the diagnostic demands, strategies have shifted toward employing techniques with higher precision, sensitivity, and better time efficiency. The integration of artificial intelligence and machine learning is set to redefine diagnostic paradigms, facilitating not only rapid and precise pathogen identification but also addressing foundational limitations in data analysis and interpretation. This review evaluates the synergy between conventional and emerging diagnostic technologies, emphasizing their clinical utility, limitations, and future trajectories for diverse audiences in microbiology and healthcare.
Acta parasitologicaMeng Yee Lai, Joe En Lim, Mohd Lutfi Abdullah, Yee Ling Lau
OBJECTIVE: The emergence of zoonotic malaria caused by Plasmodium knowlesi and P. cynomolgi presents growing challenges for malaria diagnosis in Southeast Asia. Reliable sample preservation and rapid molecular detection are particularly important in remote endemic areas where cold-chain facilities are often unavailable. Although molecular techniques provide highly sensitive and specific diagnostic tools, their application in field settings remains challenging. Consequently, FTA cards have gained increasing attention as a practical approach for the rapid collection, transport and long-term archiving of biological samples. METHODS: In this study, DNA extracted from FTA cards was evaluated using dual loop-mediated isothermal amplification (dual-LAMP) assays for parasite detection. Whole blood samples were spotted onto FTA cards, processed using a modified lysis-based protocol, and directly used as templates in dual-LAMP assays without further DNA purification. RESULTS: Both colorimetric and fluorescent LAMP assays demonstrated high specificity, with no cross-reactivity against other human malaria parasites. The assays also showed comparable analytical sensitivity, with detection limits of 1 copy/µL for P. cynomolgi and 10 copies/µL for P. knowlesi. CONCLUSION: FTA cards proved to be a faster and more cost-effective alternative to commercial DNA extraction kits. In addition, they maintained DNA stability at room temperature without the need for cold storage, making them highly suitable for field applications and reducing degradation risks during sample transportation. Combination of FTA card-based lysis and dual-LAMP provides a robust approach for malaria parasite detection.
Human geneticsSerena Redaelli, Viviana Tritto, Eleonora Mangano, Roberta Bordoni, Elena Sala, Nicoletta Villa, Francesca Crosti, Leda Dalprà, Luigina Spaccini, Alessandra Si…
Chromosomal triplications are rare structural variations often associated with complex phenotypes. We report the molecular characterization of a novel intrachromosomal triplication at 18q12.1q21.2 identified in a fetus with ultrasound abnormalities. Conventional karyotyping and array-CGH revealed a partial tetrasomy and a 26 Mb region of loss of homozygosity (LOH), extending from the triplication to the telomere. Long-read sequencing (LRS) identified breakpoint junctions revealing a direct-inverted-direct triplication structure. Breakpoint analysis suggested that this rearrangement arose through a U-type exchange between sister chromatids, likely mediated by microhomology-based mechanisms. This process likely generated a transient dicentric chromosome that subsequently broke during mitosis. The resulting duplicated chromosome may have been stabilized by telomere capture, consistent with the triplicated 18q12.1q21.2 region followed by the 18q21.2q23 LOH. Nine genes within the triplicated region, including SMAD2 and SMAD4, showed high predicted sensitivity to increased dosage, possibly contributing to the clinical phenotype. This study highlights the utility of LRS in defining complex chromosomal rearrangements and emphasizes the importance of molecular breakpoint analysis for understanding pathogenic mechanisms and improving genetic prenatal diagnosis.
The Journal of antimicrobial chemotherapyAnna Vickers, Michaela Day, Michel Doumith, Sandhya Vivekanand, Penelope R Cliff, Helen Fifer, Sarah Alexander, Rachel Pitt-Kendall
BACKGROUND: Ceftriaxone resistance threatens the last remaining empirical monotherapy for Neisseria gonorrhoeae. Ceftriaxone-resistant N. gonorrhoeae is rare in England, but cases are increasing. Rapid detection of ceftriaxone resistance, commonly conferred by mutations in the penA gene, facilitates rapid risk assessment and public health intervention including enhanced follow-up of cases. OBJECTIVES: This study aimed to develop a real-time PCR for detection of the ceftriaxone resistance-associated penA-237.001 allele in N. gonorrhoeae, for use on both cultured isolates and residual clinical specimens. METHODS: A penA-237.001-specific assay was designed and evaluated using cultured isolates, residual clinical specimens and a broad exclusivity panel. Analytical sensitivity was assessed using serial dilutions of two penA-237.001-positive isolates prepared in specimen collection kit buffers and PBS. RESULTS: The assay demonstrated 100% specificity within the validation panel; all penA-237.001-positive isolates were correctly identified with no false-positive results. Detection was reliable to approximately 15 cfu/mL across all buffer types, with minimal cycle threshold variation. CONCLUSIONS: This assay enables rapid and specific detection of penA-237.001 in cultured isolates and residual molecular specimens. Used alongside existing molecular assays, it offers enhanced early detection of ceftriaxone-resistant N. gonorrhoeae and supports timely public health action.
Frontiers in immunologyNa Jiang, Jianlin Luo, Chuiyu Kong, Zhihong Ma, Lin Luo, Xingchen Huo, Yan Chen, Huanhuan Yu, Jian Gao, Jinjing Zhang, Jinyou Hu, Dongjie Shi
INTRODUCTION: Koi herpesvirus (KHV or CyHV-3) causes a highly contagious and lethal disease in common carp and koi, resulting in substantial economic losses in global aquaculture. Rapid point-of-care testing (POCT) of KHV is critical for curbing its spread and preventing outbreaks. However, conventional diagnostic approaches, such as TaqMan quantitative PCR (qPCR), require professional personnel and specialized equipment, limiting their on-site applicability. METHODS: A rapid visual POCT assay was developed by combing loop-mediated isothermal amplification (LAMP) with non-invasive mucus swab sampling. Two optimized release solutions enabled one-step nucleic acid preparation within 5 min. Among five primer sets designed, the optimal set targeting the thymidine kinase (TK) gene was selected for isothermal amplification at 65 °C for 60 min. The assay was further adapted for naked-eye result interpretation using a pH-sensitive indicatior dye. RESULTS: The assay demonstrated a detection limit of 21.42 copies/μL, with a 95% confidence interval of 14.88-41.83 copies/μL. No cross-reactivity was observed with seven common fish pathogens. Comparative evaluation with TaqMan qPCR using parallel clinical specimens yielded 100% concordant results. The total detection time was 65 min, enabling the POCT implementation across diverse scenarios. DISCUSSION: This non-invasive, visual LAMP-based POCT platform provides a sensitive, specific and equipment-free diagnostic strategy for KHV detection, facilitating timely disease surveillance, outbreak control management, and aquaculture biosecurity.
Journal of medical virologyLinglong Wan, Juan Zhou, Lei Yu, Xiaolan Huang, Jin Fu, Fei Xiao, Nan Jia, Yu Zhang, Min Chen, Zhaomin Feng, Yi Wang
Human adenovirus (HAdV) is a leading cause of acute respiratory tract infections (ARTIs) in children. The high prevalence of HAdV serotypes 3 and 7 in regions such as China presents a significant public health challenge. Here, we propose a one-pot assay that integrates multiple cross displacement amplification (MCDA) with CRISPR-Cas12b for the detection of HAdV-3 and HAdV-7, termed HAdV-MCDA-One. In this system, MCDA provides exponential target amplification, while the collateral cleavage activity of Cas12b enables secondary signal amplification. The entire reaction is performed isothermally at 60°C in a single tube, providing a fluorescent readout within 50 min, making the assay suitable for point-of-care testing (POCT). Leveraging the single-base recognition capability of CRISPR-Cas12b, the assay demonstrates high specificity, with no cross-reactivity observed against the other 13 identified pathogens. The limit of detection was determined to be 1.59 copies per reaction using target plasmids. Moreover, when evaluated with 96 clinical pharyngeal swabs, the assay showed 100% concordance with quantitative PCR (qPCR), confirming its clinical reliability. These results demonstrate HAdV-MCDA-One as a rapid and robust tool for HAdV-3 and HAdV-7 detection, with significant potential for clinical diagnosis and public health surveillance.
Taiwanese journal of obstetrics & gynecologyYung-Ling Tseng, Ying-Chung Chen
OBJECTIVES: To present a rare prenatal case of incomplete trisomy 15 rescue with hypermethylation in the Prader-Willi critical region and to emphasize the diagnostic challenges associated with discordant results among NIPT, chromosomal microarray, and karyotype, highlighting the critical role of methylation analysis in confirming the diagnosis. CASE REPORT: Our patient of 41-yea-old underwent NIPT showed trisomy 15 but low risk in PWS and Angelman syndrome. Subsequent karyotyping and CGH array provided discordant results possibly due to incomplete trisomy rescue. To clarify the diagnosis, MLPA was performed to confirm the hypermethylation of the Prader-Willi critical region, presenting as atypical PWS. CONCLUSION: This case highlights the importance of MLPA test in the diagnosis workflow of atypical PWS caused by incomplete trisomy rescue. Early recognizing of atypical PWS is important due to its variable prognosis reported by current literatures.
Frontiers in cellular and infection microbiologyLei Yu, Bo Peng, Qingjing Du, Yangyang Wu, Shuang Liu, Chunmei Zhu, Chuanhe Liu
Respiratory syncytial virus (RSV) is a major cause of acute lower respiratory tract infections in children, highlighting the need for rapid and accessible molecular detection. Here, we developed two recombinase polymerase amplification (RPA)-based assays for the detection and differentiation of RSV-A and RSV-B, combining fluorescence readout or lateral flow biosensor (LFB) visualization, termed RSV-RPA-FLU and RSV-RPA-LFB, respectively. The workflow can be completed within 40 min, including sample preparation for 15 min, isothermal amplification for 20 min, and result interpretation within 5 min. Analytical sensitivity testing showed that the RSV-RPA-FLU assay detected both RSV-A and RSV-B at 104 copies/μL. The RSV-RPA-LFB assay showed higher sensitivity, with detection limits of 6.05 × 10³ copies/μL for RSV-A and 7.80 × 10² copies/μL for RSV-B. Specificity analysis against 11 non-RSV respiratory pathogens showed no cross-reactivity. Preliminary evaluation using simulated clinical samples further supported the feasibility of both assays in complex sample matrices. These results indicate that the RSV-RPA-FLU and RSV-RPA-LFB assays provide rapid, specific, and operationally simple analytical platforms for RSV-A/RSV-B detection and differentiation, holding great potential for point-of-care RSV diagnosis and global prevention and control.
Frontiers in immunologyBin Li, Yifei Dong, Yifei Dai, Mengpei Zhang, Zhenwei Zou, Hai Yang
Fusobacterium nucleatum (Fn) is recognized as a novel oncogenic organism closely correlated with colorectal cancer (CRC). Fn can induce gut dysbiosis and promote tumorigenesis through various mechanisms, including activation of oncogenic signaling, modulation of the immune microenvironment, metabolic reprogramming, and epigenetic regulation. Although recent technological breakthroughs have yielded high-resolution Fn detection indicators, clinical translation remains compromised by subspecies heterogeneity, emerging taxonomic updates, and non-standardization of diagnostic platforms. This review integrates recent developments regarding Fn pathogenic mechanisms and taxonomic identification in CRC, including genetic heterogeneity analysis at the species and subspecies levels, elucidation of multi-dimensional oncogenic molecular mechanisms, and application of high-resolution detection technologies. Special focus is placed on advancing these findings toward early noninvasive diagnostic markers. Additionally, we discuss the limitations of current studies, technical bottlenecks, and potential directions for future research to pave the way for standardized clinical screening.C.
Journal of microbiological methodsPromod K Mehta, Jefry Sebastian, Anshika Vashisht, Hema Saini, Amit Kumar, Abhishek Sheoran, Vipul Kumar, Raj Singh, Latika Bhayana, Mukesh Sharma, Aparna Parm…
A head-to-head comparison was made between Truenat® MTB Plus (TruPlus) vs. GeneXpert® MTB/RIF (Xpert MTB/RIF), wherein TruPlus exhibited slightly higher sensitivity (88.9% vs. 83.3%) than Xpert MTB/RIF for diagnosing pulmonary TB individuals against MGIT-960, whereas significantly higher sensitivity (p < 0.01) was obtained by TruPlus than Xpert MTB/RIF for extrapulmonary TB individuals.
Frontiers in cellular and infection microbiologyHansheng Wang, Dan Cheng, Xin Qian, Xianru Xia, Biyu Chen, Chenglin Wu, Fang Wang, Sijia Guo, Yujie Gao, Yi Wu, Tao Ren, Yijun Tang, Meifang Wang
OBJECTIVE: To evaluate the diagnostic performance of multiplex PCR-based targeted next-generation sequencing (mp-tNGS) for detecting Mycobacterium tuberculosis (MTB) in bronchoalveolar lavage fluid (BALF) from patients with suspected pulmonary tuberculosis (PTB). METHODS: This prospective study enrolled 188 patients. BALF samples were tested in parallel by AFB smear, culture, Xpert MTB/RIF, and mp-tNGS. Diagnostic performance was assessed against a composite clinical reference standard; phenotypic drug susceptibility testing (pDST) was additionally compared in a subset of 35 patients. RESULTS: Among the cohort, 116 patients were diagnosed with PTB. The sensitivities of AFB smear, culture, Xpert MTB/RIF, and mp-tNGS were 19.8%, 33.6%, 73.3%, and 83.6%, respectively; specificities were 91.7%, 100%, 98.6%, and 97.2%. mp-tNGS demonstrated the highest diagnostic accuracy (88.8%) and the strongest agreement with the reference standard (κ = 0.868, p < 0.001); Subgroup analysis using microbiological reference standard confirmed comparable performance (sensitivity 83.8%, specificity 97.2%). The area under the ROC curve for mp-tNGS was 0.88, significantly outperforming AFB smear (0.56), culture (0.67), and Xpert MTB/RIF (0.81). In the resistance subset, mp-tNGS showed 100% sensitivity and specificity for rifampicin resistance (all rpoB S450L mutations) and 75% sensitivity with 100% specificity for isoniazid resistance (katG S315T mutations), identifying five probable multidrug-resistant cases. Xpert MTB/RIF detected rifampicin resistance with 75.0% sensitivity and 96.3% specificity. CONCLUSION: The mp-tNGS assay provides a superior combination of rapid turnaround time (~12 hours) and high diagnostic accuracy for MTB detection in BALF. Beyond pathogen identification, it offers rapid and preliminary genotypic resistance screening, supporting early suspicion of multidrug resistance, while phenotypic confirmation remains essential. This makes mp-tNGS a highly effective tool for the rapid, accurate diagnosis and preliminary resistance screening of PTB.
BACKGROUND: Enteropathogenic Escherichia coli (EPEC) is a major etiological agent of persistent diarrhea in pediatric and adult population globally and it is a leading cause of infant mortality in developing countries. Bacterial culture and biochemical assays are insufficient for accurately identifying EPEC strains. In contrast, PCR serves as a major molecular diagnostic method for EPEC. However, the application of PCR is limited due to high cost, time taking and technical requirements. METHOD AND RESULTS: In this study loop mediated isothermal amplification LAMP) was developed for rapid, specific, easy and resource-friendly detection of EPEC. The LAMP assay was developed by designing specific lamp primers targeting eae genes, in combination with stx1 and stx2 lamp primers to enable EPEC detection. The reaction mixture, which contained 2.5 µL of isothermal amplification buffer, 10 mM of MgSO4 solution, 1.4 mM of dNTPs, 1.8 mM of FIP and BIP, 0.4 mM of F3 and B3, 0.2 mM of LB, 8U of Bst polymerase, and 2.0 µL of the target DNA template, was incubated at 62 C0 for 60 minutes. The designed lamp assay's performance was assessed utilizing 60 bacterial trains that were isolated locally. The assay attained 100% efficiency (60/60), 100% sensitivity (10/10), and 100% specificity (50/50). Furthermore, both the positive and negative predictive scores were 100%. Up to 0.05 pg of DNA could be detected in each reaction using the developed LAMP test. The traditional PCR, on the other hand, showed a detection limit of 5 pg/reaction. Additionally, the developed LAMP assay was able to detect up to 7 x 102 cfu/g stool in a spiked stool sample. PCR, however, can identify up to 7 x 104 cfu/g of stool. Notably, compared to conventional PCR, the LAMP assay has a 100-fold greater sensitivity. The result obtained from LAMP and PCR tests showed a perfect agreement with a kappa value of 1 (k = 1). CONCLUSION: The developed LAMP assay demonstrated promising performance for the detection of EPEC under controlled laboratory conditions, offering a rapid and straightforward alternative to conventional diagnostic methods. Clinical validation using patient stool sample and further studies with larger sample sets are needed to fully establish the assay's performance and support its broader clinical application.
Polish journal of pathology : official journal of the Polish Society of PathologistsJerzy Klijanienko
Fine needle aspiration despite being an inexpensive, rapid, accurate and non-invasive diagnostic practice, particularly when used in oncology, is frequently underutilized. The World Health Organization - International Academy of Cytology - International Agency of Research on Cancer initiated a series of Blue Books devoted solely to cytology and reporting methods in particular entities of different organs. The use of ancillary techniques such as immunocytochemistry, molecular biology and flow cytometry was defined in detail. In this review, we have focused on the role of ancillary techniques in the cytological diagnosis of soft tissue tumors. Combination of cytological and histological cellular material with ancillary techniques increases the number of accurate diagnoses and improves clinical management of patients.
Polish journal of pathology : official journal of the Polish Society of PathologistsAdam Michał Kowalewski, Łukasz Szylberg
Ovarian cancer remains the most lethal gynaecological malignancy, and it exhibits substantial histologic and molecular heterogeneity. Traditional systemic therapies have produced only modest survival gains. Integration of immunohistochemical (IHC) and molecular diagnostics has shifted care toward biomarker-driven personalised treatment. This review presents current IHC approaches for histotype classification and the most important actionable protein markers (FR, HER2, TROP2, MMR proteins, and PD-L1), together with molecular testing for BRCA1/2, homologous recombination deficiency (HRD) status, and circulating tumour DNA (ctDNA) monitoring. These diagnostics now direct poly(ADP-ribose) polymerase inhibitors maintenance in HRD-positive disease and antibody-drug conjugates such as mirvetuximab soravtansine in FR-high platinum-resistant tumours. However, important limitations remain: intratumoral heterogeneity, variable assay performance, lack of standardisation, and unequal access to testing worldwide. We also discuss how best to combine multiple biomarkers and outline future directions, such as AI-assisted digital pathology, composite predictive models, and ctDNA-guided adaptive strategies, to refine patient selection and improve long-term outcomes.
Polish journal of pathology : official journal of the Polish Society of PathologistsPavel Dundr, Michaela Kendall Bártů, Jan Hojný, Radoslav Matěj, Kristýna Němejcová
Uterine mesenchymal tumours represent a group of heterogeneous tumours, the classification of which is evolving, especially in the context of a broader use of molecular testing. Some entities can be diagnosed based on the combination of morphological and immunohistochemical features. In other entities, such as endometrial sarcomas with KAT6B/A::KANSL1 fusion, the correct diagnosis cannot currently be achieved without molecular testing. In this review, we focus on some of the new entities in which molecular testing plays an essential role for diagnosis, including tumours with KAT6B/A::KANSL1 fusion, tumours with MEIS1::NCOA2 fusion, and tumours with fusion involving the KDM2B gene. In addition, uterine tumours with whorling and GREB1:CTNNB1 fusion and endometrial stromal tumours with CTNNB1 mutation will be discussed. Other tumours with recurrent molecular alterations not specific to a single entity (such as tumours with PLAG1 fusion or RAD51B fusion) are also mentioned. In summary, molecular testing is a very important part of the classification of uterine mesenchymal tumours. The growing number of emerging entities helps to correctly classify tumours into clinically significant categories. However, these entities should be viewed with caution until their clinical significance is supported by robust data, and molecular findings should always be correlated with morphological features.
Frontiers in cellular and infection microbiologyNan Yang, Jin Zhou, Han Zhang, Yihsuan Huang, Guiping Kong, Yucan Zheng, Zhuo Zhang, Ying Shen, Xiu Han, Zhifeng Liu, Yan Lu
Diarrhea caused by bacterial and viral pathogens remains a major cause of childhood morbidity and mortality, leading to dehydration, malnutrition, and substantial healthcare burdens; rapid and accurate diagnosis is therefore essential for timely treatment. In clinical practice, detection still commonly relies on traditional methods such as stool culture and enzyme-linked immunosorbent assays. These approaches are not only time-consuming and lack sensitivity but also typically require separate, pathogen-specific tests, complicating comprehensive diagnosis. While quantitative PCR (qPCR) offers speed and sensitivity, dye-based methods lack specificity, and fluorescent probe-based assays are costly. To address these limitations, we developed an integrated assay that combines PCR with a sequence-specific invasive reaction (Invader) in a single closed tube. This method detects six major pediatric enteric pathogens: Shigella spp., Vibrio cholerae, non-typhoidal Salmonella, Campylobacter jejuni, rotavirus, and enteric adenovirus (types 40/41). The assay demonstrated high sensitivity, with detection limits as low as 2 copies per reaction for each target and exhibited no cross-reactivity. The entire process was completed within 45 minutes. By employing universal Invader probes, we reduced the cost per test to approximately 2 RMB. Validation using both simulated and clinical stool samples suggested the assay's potential reliability and consistency with standard methods. This closed-tube PCR-Invader assay provides a rapid, cost-effective, and unified platform for the parallel detection of six major diarrheal pathogens in a single test run. It shows promise as a practical alternative to current disparate and pathogen-specific testing strategies, with considerable potential for use in primary healthcare settings and large-scale screening to guide the management of childhood infectious diarrhea.