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West Nile Virus (WNV) infection represents a major global public health challenge. Even though most of the patients are asymptomatic, some cases progress to critical condition which may be fatal. Diagnosis traditionally relies on serological methods, but their limitations, including cross‐reactivity, highlight the need for alternative approaches. Here, we present the development and validation of a novel RT‐qPCR assay for precise and rapid detection of WNV RNA in urine, emerging as a promising specimen due to its noninvasive collection and high viral load. The assay demonstrates high efficiency and sensitivity, with a detection limit comparable to commercially available kits. This study highlights the importance of in‐house kit design as a diagnostic tool in regions affected by emerging tropical infections, such as WNV, exemplified Cyprus. It emphasizes the critical role of low‐cost, early detection with high sensitivity and specificity in infection control and surveillance efforts. [ABSTRACT FROM AUTHOR]

Introduction: Accurate and rapid diagnosis is crucial for the effective treatment of parasitosis. Traditional etiological methods, especially microscopic examination, are time-consuming, labor-intensive, and prone to false or missed detections. In response to these challenges, this study explores the use of artificial intelligence (AI) for the detection and classification of human parasite eggs through the YOLOv4 deep learning object detection algorithm. Methods: Eggs from species such as Ascaris lumbricoides (A. lumbricoides), Trichuris trichiura (T. trichiura), Enterobius vermicularis (E. vermicularis), Ancylostoma duodenale (A. duodenale), Schistosoma japonicum (S. japonicum), Paragonimus westermani (P. westermani), Fasciolopsis buski (F. buski), Clonorchis sinensis (C. sinensis), and Taenia spp. (T. spp.) were collected and prepared as both single species and mixed egg smears. These samples were photographed under a light microscope and analyzed using the YOLO (You Only Look Once) v4 model. Results: The model demonstrated high recognition accuracy, achieving 100% for Clonorchis sinensis and Schistosoma japonicum , with slightly lower accuracies for other species such as E. vermicularis (89.31%), F. buski (88.00%), and T. trichiura (84.85%). For mixed helminth eggs, the recognition accuracy rates arrived at Group 1 (98.10, 95.61%), Group 2 (94.86, 93.28 and 91.43%), and Group 3 (93.34 and 75.00%), indicating the platform's robustness but also highlighting areas for improvement in complex diagnostic scenarios. Discussion: The results show that this AI-assisted platform significantly reduces reliance on professional expertise while maintaining real-time efficiency and high accuracy, offering a powerful tool for the diagnosis and treatment of parasitosis. With further optimization, such as expanding training datasets and refining recognition algorithms, this AI system could become a key resource in both clinical and public health efforts to combat parasitic infections. [ABSTRACT FROM AUTHOR]

In the last few years, next-generation sequencing (NGS) has emerged as a technology for laboratory diagnosis of many culture-negative infections and slow-growing microorganisms. In this study, we describe the use of metagenomic NGS (mNGS) for rapid diagnosis of T. marneffei infection in a 37-year-old renal transplant recipient who presented with chronic pneumonia syndrome. Bronchoalveolar lavage for mNGS was positive for T. marneffei sequence reads. Prolonged incubation of the bronchoalveolar lavage revealed T. marneffei colonies after 6 days of incubation. Analysis of 23 cases of T. marneffei infections in renal transplant recipients from the literature revealed that the number of cases ranged from 1 to 4 cases per five years from 1990 to 2020; but increased rapidly to 9 cases from 2021 to 2023, with 7 of them diagnosed by NGS. Twenty of the 23 cases were from T. marneffei-endemic areas [southern part of mainland China (n = 9); Hong Kong (n = 4); northeastern India (n = 2); Indonesia (n = 1) and Taiwan (n = 4)]. For the 3 patients from non-T. marneffei-endemic areas [United Kingdom (n = 2) and Australia (n = 1)], they had travel histories to China and Vietnam respectively. The time taken for diagnosis by mNGS [median 1 (range 1 to 2) day] was significantly shorter than that for fungal culture [median 6 (range 3 to 15) days] (P = 0.002). mNGS is useful for picking up more cases of T. marneffei infections in renal transplant recipients as well as providing a rapid diagnosis. Talaromycosis is an emerging fungal infection in renal transplant recipients. [ABSTRACT FROM AUTHOR]

the aim of this study is to evaluate the accuracy of the HB&L Uroquattro instrument (Alifax, Italy) and the Residual Antimicrobial Activity test (RAA) for rapid and correct diagnosis of Urinary Tract Infections (UTIs) and to compare the results with those obtained with the classical cultural method.

Objective To establish a rapid detection method for invasive candidiasis based on droplet digital polymerase chain reaction (ddPCR). Methods We developed an assay system using a microtitre-based digital PCR platform and designed primer probes specific for four Candida species, namely Candida albicans, Candida smoothii, Candida near-smoothii, and Candida tropicalis. (1) The Limit of Blank (LOB) range and positive judgment value were determined by analyzing No Template Control (NTC) samples. (2) The Limit of Detection (LOD) range was determined by diluting positive samples with 10 replicate extractions at each concentration gradient. (3) The Linear Limit of Quantitation (LOQ) range was determined by repetitive testing of diluted samples. (4) The linear range limit was determined through gradient dilution of the positive samples. (5) The coefficient of variation (CV), calculated from the logarithmic values of the resultant concentrations, was assessed by extracting and testing positive samples in 12 repetitions at both high and low concentrations. (6) Method reliability was evaluated by calculating the CV from the logarithmic values of the resultant concentrations obtained from clinical samples with fungal culture results. Results The ddPCR assay detected Candida LOB at a range of 0 ~ 81 copies/mL, with a positive threshold set at 5 3 positive microdroplets. The LOD and LOQ were determined to be 3 x 10² copies/mL. The linear range for detecting different concentration gradients was found to be between 3 x 10² and 3 x 107 copies/mL, with high correlation coefficients observed for Candida albicans (R² = 0.999 5), Candida smoothii (R² = 0.998 9 ), Candida near-smoothii (R² = 0.999 4), and Candida tropicalis (R² = 0.999). Additionally, the coefficient of variation for the resultant concentration logarithmic values was less than 5%, meeting precision requirements. Furthermore, preliminary validation using clinical specimens demonstrated consistent results compared to clinical culture findings. Conclusion ddPCR exhibits rapidity, high sensitivity, good repeatability, and high specificity in detecting invasive candidiasis in critically ill patients. This study highlights the potential value of droplet digital PCR as a diagnostic tool for invasive candidiasis. [ABSTRACT FROM AUTHOR]

Human noroviruses (HuNoVs), the most prevalent viral contaminant in food, account for a substantial proportion of nonbacterial gastroenteritis cases. Extensive work has been focused on the diagnosis of HuNoVs in clinical samples, whereas the availability of sensitive detection methods for their detection in food is lacking. Here, we developed a virus enrichment approach utilizing graphene-based nanocomposites (CTAB-rGO-Fe3O4) that does not rely on large instruments and is suitable for on-site food pretreatment. The recovery efficiency of the developed virus enrichment procedure for serially diluted GII.4 norovirus ranged from 10.06 to 72.67% in strawberries and from 2.66 to 79.65% in oysters. Furthermore, we developed a real-time recombinase polymerase amplification (real-time RPA) assay, which can detect as low as 1.22 genome copies µL−1 of recombinant plasmid standard and has no cross-reactivity with genomes of astrovirus, rotavirus, adenovirus, and MS2 bacteriophage. Notably, the combined virus enrichment and real-time RPA detection assay enhanced the detection limits to 2.84 and 37.5 genome copies g−1 in strawberries and oysters, respectively, compared to those of qPCR. Our strategy, the graphene-based virus enrichment method combined with real-time RPA, presents a promising tool for sensitively detecting HuNoVs in food samples. [ABSTRACT FROM AUTHOR]

Mycoplasma pneumoniae is a significant pathogen responsible for communityacquired pneumonia, predominantly affecting children and adolescents. Here, we devised a rapid method for M. pneumoniae that combined multiple cross displacement amplification (MCDA) with real-time fluorescence technology. A set of ten primers, which were specifically designed for M. pneumoniae detection, were employed in a real-time fluorescence MCDA reaction. Of these, one primer incorporated a restriction endonuclease recognition sequence, a fluorophore, and a quencher, facilitating real-time fluorescence detection. The real-time (RT)-MCDA reactions were monitored in a simple realtime fluorescence instrument and conducted under optimised conditions (64°C for 40 min). The detection limit of the M. pneumoniae RT-MCDA assay for genomic DNA extracted from M. pneumoniae culture was down to 43 fg/μl. This assay accurately identified M. pneumoniae strains without cross-reacting with other bacteria. To validate its practical application, we tested the M. pneumoniae RT-MCDA assay using genomic DNA extracted from clinical samples. The assay's detection capability proved comparable with real-time PCR, MCDA-based biosensor detection, and visual inspection under blue light. The entire process, including rapid DNA extraction and real-time MCDA detection, was completed within 1 h. Overall, the M. pneumoniae RT-MCDA assay reported here is a simple and effective diagnostic tool for rapid M. pneumoniae detection, which holds significant potential for point-of-care testing and in resource-limited regions. [ABSTRACT FROM AUTHOR]

IntroductionAccurate and rapid diagnosis is crucial for the effective treatment of parasitosis. Traditional etiological methods, especially microscopic examination, are time-consuming, labor-intensive, and prone to false or missed detections. In response to these challenges, this study explores the use of artificial intelligence (AI) for the detection and classification of human parasite eggs through the YOLOv4 deep learning object detection algorithm.MethodsEggs from species such as Ascaris lumbricoides (A. lumbricoides), Trichuris trichiura (T. trichiura), Enterobius vermicularis (E. vermicularis), Ancylostoma duodenale (A. duodenale), Schistosoma japonicum (S. japonicum), Paragonimus westermani (P. westermani), Fasciolopsis buski (F. buski), Clonorchis sinensis (C. sinensis), and Taenia spp. (T. spp.) were collected and prepared as both single species and mixed egg smears. These samples were photographed under a light microscope and analyzed using the YOLO (You Only Look Once) v4 model.ResultsThe model demonstrated high recognition accuracy, achieving 100% for Clonorchis sinensis and Schistosoma japonicum, with slightly lower accuracies for other species such as E. vermicularis (89.31%), F. buski (88.00%), and T. trichiura (84.85%). For mixed helminth eggs, the recognition accuracy rates arrived at Group 1 (98.10, 95.61%), Group 2 (94.86, 93.28 and 91.43%), and Group 3 (93.34 and 75.00%), indicating the platform’s robustness but also highlighting areas for improvement in complex diagnostic scenarios.DiscussionThe results show that this AI-assisted platform significantly reduces reliance on professional expertise while maintaining real-time efficiency and high accuracy, offering a powerful tool for the diagnosis and treatment of parasitosis. With further optimization, such as expanding training datasets and refining recognition algorithms, this AI system could become a key resource in both clinical and public health efforts to combat parasitic infections.

Abstract Background Senecavirus A (SVA), identified in 2002, is known to cause porcine idiopathic vesicular disease (PIVD), which presents with symptoms resembling other vesicular diseases. This similarity complicates field diagnosis. Conventional molecular diagnostic techniques are limited by their cost, sensitivity, and requirement for complicated instrumentation. Therefore, developing an effective and accurate diagnostic method is crucial for timely identification and isolation of affected pigs, thereby preventing further disease spread. Methods In this study, we developed a highly-specific and ultra-sensitive SVA detection method powered by CRISPR/Cas12a. To enhance the availability in laboratories with varied equipment conditions, microplate reader and ultraviolet light transilluminator were introduced. Moreover, PCR amplification has also been incorporated into this method to improve sensitivity. The specificity and sensitivity of this method were determined following the preparation of the recombinant Cas12a protein and optimization of the CRISPR/Cas12a-based trans-cleavage system. Results The method demonstrated no cross-reactivity with ten kinds of viruses of swine. The minimum template concentration required to activate substantial trans-cleavage activity was determined to be 106 copies/µL of SVA templates. However, when PCR amplification was incorporated, the method achieved a detection limit of one copy of SVA templates per reaction. It also exhibited 100% accuracy in simulated sample testing. The complete testing process does not exceed three hours. Conclusions Importantly, this method utilizes standard laboratory equipment, making it accessible for use in resource-limited settings and facilitating widespread and ultra-sensitive screening during epidemics. Overall, the development of this method not only broadens the array of tools available for detecting SVA but also holds significant promise for controlling the spread of PIVD.

Abstract Cassava brown streak disease (CBSD) caused by Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV) is the most economically important viral disease of cassava. As cassava is a vegetatively propagated crop, the development of rapid and sensitive diagnostics would aid in the identification of virus-free planting material and development of effective management strategies. In this study, a rapid, specific and sensitive real-time reverse transcription recombinase polymerase amplification (RT-RPA) assay was developed for real-time detection of CBSV and UCBSV. The RT-RPA was able to detect as little as 2 pg/µl of purified RNA obtained from infected cassava leaves, a sensitivity equivalent to that obtained by quantitative real-time reverse transcription PCR (qRT-PCR), within 20 min at 37 °C. Further, the RT-RPA detected each target virus directly from crude leaf and stem extracts, avoiding the tedious and costly isolation of high-quality RNA. The developed RT-RPA assay provides a valuable diagnostic tool that can be adopted by cassava seed certification and virus resistance breeding programs to ensure distribution of virus-free cassava planting materials to farmers. This is the first report on the development and validation of crude sap-based RT-RPA assay for the detection of cassava brown streak viruses (UCBSV and CBSV) infection in cassava plants.

Pathogenic microorganisms play a crucial role in the global disease burden due to their ability to cause various diseases and spread through multiple transmission routes. Immunity tests identify antigens related to these pathogens, thereby confirming past infections and monitoring the host's immune response. Traditional pathogen detection methods, including enzyme-linked immunosorbent assays (ELISAs) and chemiluminescent immunoassays (CLIAs), are often labor-intensive, slow, and reliant on sophisticated equipment and skilled personnel, which can be limiting in resource-poor settings. In contrast, the development of microfluidic technologies presents a promising alternative, offering automation, miniaturization, and cost efficiency. These advanced methods are poised to replace traditional assays by streamlining processes and enabling rapid, high-throughput immunity testing for pathogens. This review highlights the latest advancements in microfluidic systems designed for rapid and high-throughput immunity testing, incorporating immunosensors, single molecule arrays (Simoas), a lateral flow assay (LFA), and smartphone integration. It focuses on key pathogenic microorganisms such as SARS-CoV-2, influenza, and the ZIKA virus (ZIKV). Additionally, the review discusses the challenges, commercialization prospects, and future directions to advance microfluidic systems for infectious disease detection. [ABSTRACT FROM AUTHOR]

During their development, some species of salmonids may occasionally experience skeletal deformations. Several methodologies are currently being used for the diagnosis of such malformations, among which X-rays, histological techniques, diaphanization coupled either with Alizarin Red or Alcian Blue stains, as well as Scanning Electron Microscopy (SEM) can be mentioned. Each one of those methods presents inherent advantages and disadvantages. The purpose of this study was twofold: Firstly, to evaluate and compare the effectiveness of microcomputed tomography (Micro-CT) technology for anatomical analysis, three-dimensionally reconstructing the obtained images; and secondly, to contrast those images with the results obtained through the diaphanization technique. The caudal fins of five specimens of the Oncorhynchus kisutch salmon were analyzed: Two specimens were subjected to diaphanization and three were processed for Micro-CT analysis, using the BRUKER SkyScan 1272 equipment. The Micro-CT technology demonstrated superiority in the resolution of bone structures, facilitating a detailed exploration of morphological variations, as well as the distribution of mineral density. This experimental approach allowed us to identify anomalies in the morphology and growth of the last vertebrae and dorsal lepidotrichiae, as well as an increased mineral density in the malformed dorsal lepidotrichiae. The higher resolution provided by Micro-CT not only enhances our understanding of the fish ontogeny and its adaptation to diverse environments, but also opens innovative perspectives for the study of the evolution of locomotor strategies and adaptive responses to environmental changes. [ABSTRACT FROM AUTHOR]

AbstractHelicobacter pylori is a gram-negative bacterium that colonizes the stomach of approximately half of the worldwide population, with higher prevalence in densely populated areas like Asia, the Caribbean, Latin America, and Africa. H. pylori infections range from asymptomatic cases to potentially fatal diseases, including peptic ulcers, chronic gastritis, and stomach adenocarcinoma. The management of these conditions has become more difficult due to the rising prevalence of drug-resistant H. pylori infections, which ultimately lead to gastric cancer and mucosa-associated lymphoid tissue (MALT) lymphoma. In 1994, the International Agency for Research on Cancer (IARC) categorized H. pylori as a Group I carcinogen, contributing to approximately 780,000 cancer cases annually. Antibiotic resistance against drugs used to treat H. pylori infections ranges between 15% and 50% worldwide, with Asian countries having exceptionally high rates. This review systematically examines the impacts of H. pylori infection, the increasing prevalence of antibiotic resistance, and the urgent need for accurate diagnosis and precision treatment. The present status of precision treatment strategies and prospective approaches for eradicating infections caused by antibiotic-resistant H. pylori will also be evaluated. [ABSTRACT FROM AUTHOR]

Background: Senecavirus A (SVA), identified in 2002, is known to cause porcine idiopathic vesicular disease (PIVD), which presents with symptoms resembling other vesicular diseases. This similarity complicates field diagnosis. Conventional molecular diagnostic techniques are limited by their cost, sensitivity, and requirement for complicated instrumentation. Therefore, developing an effective and accurate diagnostic method is crucial for timely identification and isolation of affected pigs, thereby preventing further disease spread. Methods: In this study, we developed a highly-specific and ultra-sensitive SVA detection method powered by CRISPR/Cas12a. To enhance the availability in laboratories with varied equipment conditions, microplate reader and ultraviolet light transilluminator were introduced. Moreover, PCR amplification has also been incorporated into this method to improve sensitivity. The specificity and sensitivity of this method were determined following the preparation of the recombinant Cas12a protein and optimization of the CRISPR/Cas12a-based trans-cleavage system. Results: The method demonstrated no cross-reactivity with ten kinds of viruses of swine. The minimum template concentration required to activate substantial trans-cleavage activity was determined to be 106 copies/µL of SVA templates. However, when PCR amplification was incorporated, the method achieved a detection limit of one copy of SVA templates per reaction. It also exhibited 100% accuracy in simulated sample testing. The complete testing process does not exceed three hours. Conclusions: Importantly, this method utilizes standard laboratory equipment, making it accessible for use in resource-limited settings and facilitating widespread and ultra-sensitive screening during epidemics. Overall, the development of this method not only broadens the array of tools available for detecting SVA but also holds significant promise for controlling the spread of PIVD. [ABSTRACT FROM AUTHOR]

Within the fields of infectious disease diagnostics, microfluidic-based integrated technology systems have become a vital technology in enhancing the rapidity, accuracy, and portability of pathogen detection. These systems synergize microfluidic techniques with advanced molecular biology methods, including reverse transcription polymerase chain reaction (RT-PCR), loop-mediated isothermal amplification (LAMP), and clustered regularly interspaced short palindromic repeats (CRISPR), have been successfully used to identify a diverse array of pathogens, including COVID-19, Ebola, Zika, and dengue fever. This review outlines the advances in pathogen detection, attributing them to the integration of microfluidic technology with traditional molecular biology methods and smartphone- and paper-based diagnostic assays. The cutting-edge diagnostic technologies are of critical importance for disease prevention and epidemic surveillance. Looking ahead, research is expected to focus on increasing detection sensitivity, streamlining testing processes, reducing costs, and enhancing the capability for remote data sharing. These improvements aim to achieve broader coverage and quicker response mechanisms, thereby constructing a more robust defense for global public health security. [ABSTRACT FROM AUTHOR]

Background: Acute promyelocytic leukemia (APL) is a specific type of acute myeloid leukemia [1,2], the onset of the disease is insidious and the disease progresses rapidly, and failure to detect it in time or missing the best time to seek medical treatment is likely to cause secondary cerebral hemorrhage and lead to early death (ED: deaths occur in the first 30 days post diagnosis) [3-5]. Methods: A patient with APL was rapidly identified by peripheral blood image, fibrinogen (FIB), and D-dimer within 24 hours. Finally, APL was confirmed by bone marrow cell morphology, molecular biology, and cytogenetics. Results: The presence of faggot cells with Auer rods in the peripheral blood image and the coagulation function changes abnormally at the same time. Once the above abnormal results are found, APL should be highly suspected and timely reported to the clinic for corresponding treatment. Conclusions: APL is a critical disease, the time limit for definitive diagnosis should be calculated in hours rather than days. Peripheral blood smear microscopic examination can effectively screen out rare promyelocytes and combine with abnormal FIB and D-dimer results that are highly suspicious of APL. These methods have important clinical significance in the initial screening, early diagnosis, and reduction of early mortality due to APL. [ABSTRACT FROM AUTHOR]

Cassava brown streak disease (CBSD) caused by Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV) is the most economically important viral disease of cassava. As cassava is a vegetatively propagated crop, the development of rapid and sensitive diagnostics would aid in the identification of virus-free planting material and development of effective management strategies. In this study, a rapid, specific and sensitive real-time reverse transcription recombinase polymerase amplification (RT-RPA) assay was developed for real-time detection of CBSV and UCBSV. The RT-RPA was able to detect as little as 2 pg/µl of purified RNA obtained from infected cassava leaves, a sensitivity equivalent to that obtained by quantitative real-time reverse transcription PCR (qRT-PCR), within 20 min at 37 °C. Further, the RT-RPA detected each target virus directly from crude leaf and stem extracts, avoiding the tedious and costly isolation of high-quality RNA. The developed RT-RPA assay provides a valuable diagnostic tool that can be adopted by cassava seed certification and virus resistance breeding programs to ensure distribution of virus-free cassava planting materials to farmers. This is the first report on the development and validation of crude sap-based RT-RPA assay for the detection of cassava brown streak viruses (UCBSV and CBSV) infection in cassava plants. [ABSTRACT FROM AUTHOR]

Familial hemophagocytic lymphohistiocytosis (FHLH) is a fatal hyperinflammation syndrome arising from the genetic defect of perforin-mediated cytolysis. Curative hematopoietic cell transplantation (HCT) is needed before development of central nervous system (CNS) disease. We studied treatment outcomes of 13 patients (FHLH2 n = 11, FHLH3 n = 2) consecutively diagnosed from 2011 to 2022 by flow cytometric screening for non-myeloablative HCT in a regional treatment network in Kyushu, Japan. One patient with a novel PRF1 variant escaped screening, but all patients with FHLH2 reached diagnosis and 8 of them received HCT until 3 and 9 months of age, respectively. The earliest HCT was conducted 65 days after birth. Three pretransplant deaths occurred in newborns with liver failure at diagnosis. Ten posttransplant patients have remained disease-free, 7 of whom had no neurological involvement. Time from first etoposide infusion to HCT was shorter in patients without CNS disease or bleeding than in patients with those factors (median [range] days: 62 [50–81] vs. 122 [89–209], p = 0.016). Six of 9 unrelated patients had a PRF1 c.1090_1091delCT variant. These results suggest that the critical times to start etoposide and HCT are within 3 months after birth and during etoposide control, respectively. Newborn screening may increase the percentage of disease-free survivors without complications. [ABSTRACT FROM AUTHOR]

ABSTRACT Equine piroplasmosis (EP) is a protozoal disease affecting equids, caused by Theileria equi and Babesia caballi. EP is conventionally diagnosed using microscopic, molecular, and/or serological methods, which are time-consuming. Consequently, there is a need for faster testing methods. In this study, we evaluated the application of the Sysmex XN-31 automated hematology analyzer, originally a rapid test for detecting malaria in humans, for the diagnosis of EP. The cultured parasites were measured using the XN-31 that had been customized for horse blood samples (XN-31m). The following parameters were evaluated: limit of detection (LoD), limit of quantification (LoQ), linearity, carryover, precision, and correlation with microscopic examination. The XN-31m detected infected red blood cells (RBCs) in approximately 1 minute. The LoD and LoQ for B. caballi were 4.54 infected RBCs/μL and 14.10 infected RBCs/μL, while those for T. equi were 5.80 infected RBCs/μL and 11.44 infected RBCs/μL, respectively. Linearity showed excellent correlation (R2 > 0.99), and carryover never exceeded 0.5%. The coefficient of variation was under 5%. The correlation between the results obtained using XN-31m and microscopic examination was high (R2 > 0.98). In conclusion, the XN-31 analyzer detected B. caballi and T. equi parasites in approximately 1 minute with high sensitivity. The results indicate the potential of the XN-31 analyzer as a fast and user-friendly diagnostic method for EP.IMPORTANCEIn this study, we demonstrated that the automated hematology analyzer, XN-31, can detect red blood cells infected with Babesia caballi and Theileria equi in about 1 minute. We evaluated the diagnostic performance of the XN-31 analyzer for equine piroplasmosis, providing evidence of its potential as a diagnostic tool for this disease.

Heat shock protein 70(HSP70) is a protein that can inhibit cell protein denaturation or expansion under stress or high temperature. It plays a key role in the expression of inflammatory factors, inhibition of apoptosis, and inhibition of protein aggregation. HSP70 is a protein that can inhibit cell protein denaturation or expansion under stress or high temperature. It plays a key role in the expression of inflammatory factors, inhibition of apoptosis, and inhibition of protein aggregation. This paper expounds the close relationship between heat shock protein 70 and the pathogenesis of lung diseases such as lung cancer, pneumonia, pulmonary tuberculosis and asthma, and provides a new idea for the rapid diagnosis of lung diseases. [ABSTRACT FROM AUTHOR]

Edwardsiella piscicida, a significant intracellular pathogen, is widely distributed in aquatic environments and causes systemic infection in various species. Therefore, it's essential to develop a rapid, uncomplicated and sensitive method for detection of E. piscicida in order to control the transmission of this pathogen effectively. The recombinase-aided amplification (RAA) assay is a newly developed, rapid detection method that has been utilized for various pathogens. In the present study, a real-time RAA (RT-RAA) assay, targeting the conserved positions of the EvpP gene, was successfully established for the detection of E. piscicida. This assay can be performed in a one-step single tube reaction at a temperature of 39°C within 20 min. The RT-RAA assay exhibited a sensitivity of 42 copies per reaction at a 95% probability, which was comparable to the sensitivity of real-time quantitative PCR (qPCR) assay. The specificity assay confirmed that the RT-RAA assay specifically targeted E. piscicida without any cross-reactivity with other important marine bacterial pathogens. Moreover, when clinical specimens were utilized, a perfect agreement of 100% was achieved between the RT-RAA and qPCR assays, resulting a kappa value of 1. These findings indicated that the established RT-RAA assay provided a viable alternative for the rapid, sensitive, and specific detection of E. piscicida. [ABSTRACT FROM AUTHOR]

Bakanae disease is a rice seedborne disease caused by the Fusarium (Gibberella) fujikuroi species complex (FFSC), among which F. fujikuroi is the dominant pathogen. Pathogens usually hide inside or on the surface of seeds, and infection occurs mainly at the germination stage. In this study, a method for the detection of F. fujikuroi in rice seeds and seed soaking water samples was established using recombinase polymerase amplification (RPA) technology with lateral flow device (LFD) chromatography test strips. A pair of specific primers and probes based on the cyp51c gene were screened. RPA‐LFD was used to detect 10 F. fujikuroi strains, and the results showed that all of them tested positive and there was no cross‐reaction with other Fusarium or non‐Fusarium species. The target production of the RPA‐LFD assay was obtained at 35–45°C for 8–14 min, and optimal reaction conditions of amplification at 39°C for 8 min is recommended. The sensitivity test showed that the detection limit of the RPA‐LFD test for F. fujikuroi genomic DNA in rice seeds was 100 fg/μL, and the detection limit for F. fujikuroi spores in submerged water samples was 100 spores/mL. In the assay for field samples, it successfully detected F. fujikuroi carried in the seeds of three out of five rice varieties. In addition, the whole RPA‐LFD assay can specifically detect F. fujikuroi within 30 min. This method is expected to become an early field monitoring tool for rice bakanae disease. [ABSTRACT FROM AUTHOR]

The aim of this study was to develop a multiplex PCR assay capable of rapidly differentiating two major Avipoxvirus (APV) species, Fowlpox virus (FWPV) and Pigeonpox virus (PGPV), which cause disease in bird species. Despite the importance of a rapid differentiation assay, no such assay exists that can differentiate the APV species without sequencing. To achieve this, species-specific target DNA fragments were selected from the fpv122 gene of FWPV and the HM89_gp120 gene of PGPV, which are unique to each genome. Nine samples collected from unvaccinated chickens, pigeons, and a turkey with typical pox lesions were genetically identified as FWPV and PGPV. The designed primers and target DNA fragments were validated using in silico analyses with the nucleotide Basic Local Alignment Search Tool. The multiplex PCR assay consisted of species-specific primers and previously described PanAPV primers (genus-specific) and was able to differentiate FWPV and PGPV, consistent with the phylogenetic outputs. This study represents the first successful differentiation of FWPV and PGPV genomes using a conventional multiplex PCR test. This assay has the potential to facilitate the rapid diagnosis and control of APV infections. Desarrollo de un ensayo de PCR múltiple para la diferenciación rápida de los virus de la viruela aviar y la viruela de paloma. El objetivo de este estudio fue desarrollar un ensayo de PCR múltiple capaz de diferenciar rápidamente dos especies principales de Avipoxvirus (APV) (viruela del pollo), el Fowlpox virus (FWPV) y el Pigeonpox virus (PGPV), (viruela de la gallina), que causan enfermedades en especies de aves. A pesar de la importancia de un ensayo de diferenciación rápida, no existe ningún ensayo que pueda diferenciar las especies de APV sin secuenciación. Para lograr esto, se seleccionaron fragmentos blanco de ADN específicos de especie del gene fpv122 de FWPV y el gene HM89_gp120 de Pigeonpox virus, que son únicos para cada genoma. Nueve muestras recolectadas de pollos, palomas y un pavo que no fueron vacunados con lesiones típicas de la viruela se identificaron genéticamente como FWPV y PGPV. Los iniciadores diseñados y los fragmentos de ADN blanco se validaron mediante análisis in silico mediante la herramienta de búsqueda de alineación local básica de nucleótidos (BLAST). El ensayo de PCR múltiple consistió en iniciadores específicos de especie y cebadores PanAPV previamente descritos (específicos de género) y fue capaz de diferenciar entre Fowlpox virus y Pigeonpox virus, de acuerdo con los resultados filogenéticos. Este estudio representa la primera diferenciación exitosa de los genomas de Fowlpox virus y Pigeonpox virus utilizando una prueba de PCR múltiple convencional. Este ensayo tiene el potencial de facilitar el diagnóstico rápido y el control de las infecciones por Avipoxvirus. [ABSTRACT FROM AUTHOR]

Molecular techniques of nucleic acid testing recommended by the World Health Organization (WHO) for the Mycobacterium tuberculosis (MTB) detection were considered to have the potential access to the accurate tuberculosis (TB) notifications. In this study, a new method, which coupled real-time (rt) fluorescence technique with multiple cross displacement amplification (MCDA), was developed for the rapid, sensitive and specific detection of MTB (termed MTB-rt-MCDA). According to the principle of the rt-MCDA test, a set of ten primers were designed for the MCDA reaction, of which one was engineered with a restrictive endonuclease recognition site, a fluorophore and a quencher for achieving the real-time fluorescence detection. MTB-rt-MCDA test was conducted under the optimized conditions (67 °C, 40 min) on the real-time fluorescence platform. The MTB-rt-MCDA assay accurately identified the MTB strains with no cross reaction with other bacteria. The lowest detectable genomic DNA concentration of the MTB-rt-MCDA assay was 25 fg/μl. We employed the genomic DNA templates extracted from sputum of clinical cases for validating the practical applicability of this assay, and the detection power of the MTB-rt-MCDA assay was comparable to that of the Xpert method and MCDA-based biosensor detection and superior to smear microscope method. The complete process of the MTB-rt-MCDA assay, including rapid extraction of DNA and rt-MCDA test, takes less than 1 h. In conclusion, the presented MTB-rt-MCDA assay provided an effective and simple option for the rapid screening of MTB infection.

Background: The second wave of the coronavirus disease 2019 (COVID-19) pandemic recorded a surge in rhino-orbital-cerebral mucormycosis (ROCM) infection in COVID-19-positive patients with diabetes and on concomitant steroid therapy. The rapidly progressive and devastating nature of the disease necessitated prompt diagnosis and early intervention to improve patient outcomes. Histopathology and fungal culture remain essential tools; however, these investigations have long and variable turn-around times (TATs) and may delay the initiation of treatment. Frozen section is not widely available and should be avoided in COVID-19-positive cases due to the risk of aerosol production and droplet exposure. In cases with high clinicoradiologic suspicion for mucormycosis, imprint cytologic evaluation provides a rapid diagnosis. Familiarity with fungal cytomorphology, awareness of morphologic pitfalls, and implementation of a standardized reporting format aid in diagnostic accuracy. Method: Eighteen COVID-19-positive patients, who were admitted to our hospital with clinical suspicion of mucormycosis during June and July 2021, were included in the study. We used nasal or oral imprint cytology for the initial, rapid detection of Mucor. Cytology findings were correlated with histopathology and fungal culture results. Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were calculated. Results: The sensitivity, specificity, PPV, and NPV were 100%, 100%, 100% and 100%, respectively. Conclusion: This study showed that imprint cytology can be a rapid, cost-effective, first-line diagnostic modality in Mucor diagnosis.

AbstractHepatitis A virus (HAV) is mainly transmitted via contaminated food and water. HAV infection is a major global public health problem. Thus, developing a simple, rapid detection method is crucial for containing HAV epidemics, particularly in developing regions with limited laboratory resources. This study established a feasible HAV detection solution by combining reverse transcription multienzyme isothermal rapid amplification (RT-MIRA) and lateral flow dipstick (LFD) strips. Primers targeting the conserved 5’UTR sequence of HAV were used in the RT-MIRA-LFD assay. RNA extraction was enhanced by obtaining RNA directly from the centrifuged supernatant. Our study found that MIRA amplification could be finished in 12 min at 37 °C and naked-eye observation of the LFD strips in 10 min. The detection sensitivity of this method reached 1 copy/μl. RT-MIRA-LFD was compared to conventional RT-PCR using 35 human blood samples. The accuracy of the RT-MIRA-LFD method was 100%. The convenience, sensitivity, and rapidness of this detection method could provide a considerable advantage for diagnosing and controlling HAV infection, especially in regions with limited medical resources.

Deyong Tan,1– 4 Jianfeng Han,5 Qingzhi Sun,5 Xing Cheng,5 Juan Liu,5 Jia Liu,5 Qing Li,1– 4 Lizhong Dai5 1Department of Clinical Pharmacology, Xiangya Hospital, Central South University, Changsha, 410008 People’s Republic of China; 2Institute of Clinical Pharmacology, Central South University, Hunan Key Laboratory of Pharmacogenetics, Changsha, 410078 People’s Republic of China; 3Engineering Research Center of Applied Technology of Pharmacogenomics, Ministry of Education, Changsha, 410078 People’s Republic of China; 4National Clinical Research Center for Geriatric Disorders, Changsha, Hunan, 410008 People’s Republic of China; 5Sansure Biotech Inc, Changsha, Hunan Province, People’s Republic of ChinaCorrespondence: Qing Li, Department of Clinical Pharmacology, Xiangya Hospital, Central South University, 87 Xiangya Road, Changsha, Hunan, People’s Republic of China, Tel +86 731 84805380, Fax +86 731-82354476, Email liqing9251026@csu.edu.cn Lizhong Dai, Sansure Biotech Inc, No. 680 Lusong Road, Changsha, Hunan, People’s Republic of China, Tel +86 73188883176, Email lizhongd@sansure.com.cnObjective: In this study, we conducted a multi-center research on six common lower respiratory tract pathogens using novel multiplex fluorescence quantitative polymerase chain reaction (PCR), and investigated the additional diagnostic value of this method, to provide a molecular diagnostic basis for clinical practice.Methods: From March 2019 to October 2021, a total of 2047 respiratory sputum samples were collected from Hunan Provincial People’s Hospital (the First Affiliated Hospital of Hunan Normal University), Hunan Provincial Children’s Hospital, Jiangxi Provincial Children’s Hospital, and Wuhan Infectious Disease Hospital. The samples were analyzed using a novel multiplex fluorescence quantitative PCR method for Klebsiella pneumoniae, Streptococcus pneumoniae, Haemophilus influenzae, Pseudomonas aeruginosa, Legionella pneumophila, and Staphylococcus aureus. The results were compared to the results of bacterial culture and sequencing, as well as the results of third-party kits.Results: Compared to the bacterial culture method, 2047 samples were detected with a sensitivity of 100%, a specificity of 72.22%, and an overall compliance rate of 81.91%. Compared to the sequencing method, the positive agreement percentage was 99.88%, the negative agreement percentage was 97.72%, and the overall agreement rate was 98.84%. Compared to similar control reagents, the positive agreement percentage was 100%, negative agreement percentage was 79.79%, and overall compliance rate was 96.19%.Conclusion: The multiplex fluorescence PCR method has the advantages of simultaneously detecting multiple pathogenic bacteria and reducing the duration of pathogen culture identification. Combined detection can increase the detection rate, which has favorable performance and application prospects.Keywords: bacterial infection, lower respiratory tract, melting curve, multiplex PCR, rapid diagnosis

Introduction: The aim of this study was to evaluate the performance of the Xpert MTB/RIF test in the diagnosis of M. tuberculosis in pulmonary and extrapulmonary clinical specimens with positive or negative smears. Materials and Methods: Between January 2018 and December 2021, a total of 2082 samples were examined, including 1526 respiratory samples and 556 non-respiratory samples. The samples processed for culture were inoculated into Lowenstein Jensen (LJ) medium and Mycobacteria Growth Indicator Tube (MGIT) tubes, then MGITs were loaded into the MGIT 960 automated system. The Xpert MTB/RIF molecular test was performed to all samples according to the manufacturer's recommendations. Results: M. tuberculosis was grown in culture in 153 (7,3%) of all samples, and the Xpert MTB/RIF test was positive in 203 (9,7%). ARB, MTB/RIF test and culture positivity in lung samples are; 86 (5,6%), 175 (11,4%) and 129 (8,4%), respectively. ​In extrapulmonary samples; the positivity was 7 (1,2%), 28 (5%) and 24 (4,3%). When mycobacterial culture results are accepted as reference, the sensitivity was 53,6% and the specificity was 99,4% for the EZN staining method. For respiratory samples, these values ​​were 58,1% and 99,2%; for extrapulmonary samples, sensitivity and specificity were 29,2% and 100%, respectively. For all the samples examined with The Xpert MTB/RIF test; sensitivity, specificity; positive predictive value (PPV); and negative predictive value (NPV) were calculated as 89,5%, 96,6%, 67,5% and 99,1%, respectively. Conclusion: The sensitivity and specificity rates of the Xpert MTB/RIF test used in this study in non-respiratory samples were found to be slightly lower than in respiratory samples. It had high sensitivity and specificity rates in both sample groups. It was observed that The Xpert MTB/RIF test was a very fast and requiring low workload.

Human noroviruses (HuNoVs), the most prevalent viral contaminant in food, account for a substantial proportion of nonbacterial gastroenteritis cases. Extensive work has been focused on the diagnosis of HuNoVs in clinical samples, whereas the availability of sensitive detection methods for their detection in food is lacking. Here, we developed a virus enrichment approach utilizing graphene-based nanocomposites (CTAB-rGO-Fe3O4) that does not rely on large instruments and is suitable for on-site food pretreatment. The recovery efficiency of the developed virus enrichment procedure for serially diluted GII.4 norovirus ranged from 10.06 to 72.67% in strawberries and from 2.66 to 79.65% in oysters. Furthermore, we developed a real-time recombinase polymerase amplification (real-time RPA) assay, which can detect as low as 1.22 genome copies µL−1 of recombinant plasmid standard and has no cross-reactivity with genomes of astrovirus, rotavirus, adenovirus, and MS2 bacteriophage. Notably, the combined virus enrichment and real-time RPA detection assay enhanced the detection limits to 2.84 and 37.5 genome copies g−1 in strawberries and oysters, respectively, compared to those of qPCR. Our strategy, the graphene-based virus enrichment method combined with real-time RPA, presents a promising tool for sensitively detecting HuNoVs in food samples.

Acute bacterial meningitis (ABM) is a life threatening infection in children, associated with long term complications and high mortality rate.1,2 Gram staining and culture are routinely used for diagnosis of ABM. Antigen detection by latex agglutination can provide prompt results thereby facilitating early initiation of empirical antibiotic treatment. To estimate the proportion of Laboratory confirmed cases among children admitted with clinical suspicion of acute bacterial meningitis in a tertiary care hospital. To compare and analyse the diagnostic efficacy of Culture, Gram stain and antigen detection by Latex agglutination in Cerebrospinal fluid (CSF) samples for laboratory detection of Acute bacterial meningitis. CSF samples from pediatric patients with clinical suspicion of ABM were analysed by Gram stain, culture and Antigen detection by Latex agglutination method. Results were recorded and analysed. Of the 50 clinically suspected cases, 13(26%) were confirmed as Acute bacterial meningitis by laboratory investigations. Among the organisms identified, Streptococcus pneumoniae was the most common isolate in 5(38.46%) cases followed by Neisseria meningitidis, Klebsiella pneumoniae and Acinetobacter baumannii in 2(15.38%) cases each and Escherichia coli and Group B Streptococcus in 1(7.69%) case each. Among the confirmed cases, 7(53%) samples showed culture positivity while Gram stain identified 8(61.53%)cases. Latex agglutination test showed positivity in 9(69.23%) cases. In life threatening infections like acute bacterial meningitis, where early diagnosis and prompt treatment is of utmost importance, Latex agglutination test can provide results within minutes facilitating early initiation of empirical therapy, making it an effective adjunct to gram stain and culture.

Abstract Background No univocal recommendation exists for microbiological diagnosis of ventilator-associated pneumonia (VAP). Sampling of either proximal or distal respiratory tract likely impacts on the broad range of VAP incidence between cohorts. Immune biomarkers to rule-in/rule-out VAP diagnosis, although promising, have not yet been validated. COVID-19-induced ARDS made VAP recognition even more challenging, often leading to overdiagnosis and overtreatment. We evaluated the impact of different respiratory samples and laboratory techniques on VAP incidence and microbiological findings in COVID-19 patients. Methods Prospective single-centre cohort study conducted among COVID-19 mechanically ventilated patients in Policlinico Hospital (Milan, Italy) from January 2021 to May 2022. Microbiological confirmation of suspected VAP (sVAP) was based on concomitant endotracheal aspirates (ETA) and bronchoalveolar lavage (BAL). Conventional and fast microbiology (FILMARRAY® Pneumonia Panel plus, BALFAPPP) as well as immunological markers (immune cells and inflammatory cytokines) was analysed. Results Seventy-nine patients were included. Exposure to antibiotics and steroid therapy before ICU admission occurred in 51/79 (64.6%) and 60/79 (65.9%) patients, respectively. Median duration of MV at VAP suspicion was 6 (5–9) days. Incidence rate of microbiologically confirmed VAP was 33.1 (95% CI 22.1–44.0) and 20.1 (95% CI 12.5–27.7) according to ETA and BAL, respectively. Concordance between ETA and BAL was observed in 35/49 (71.4%) cases, concordance between BALFAPPP and BAL in 39/49 (79.6%) cases. With BAL as reference standard, ETA showed 88.9% (95% CI 70.8–97.7) sensitivity and 50.0% (95% CI 28.2–71.8) specificity (Cohen’s Kappa 0.40, 95% CI 0.16–0.65). BALFAPPP showed 95.0% (95% CI 75.1–99.9) sensitivity and 69% (95% CI 49.2–84.7) specificity (Cohen’s Kappa 0.60, 95% CI 0.39–0.81). BAL IL-1β differed significantly between VAP (135 (IQR 11–450) pg/ml) and no-VAP (10 (IQR 2.9–105) pg/ml) patients (P = 0.03). Conclusions In COVID-19 ICU patients, differences in microbial sampling at VAP suspicion could lead to high variability in VAP incidence and microbiological findings. Concordance between ETA and BAL was mainly limited by over 20% of ETA positive and BAL negative samples, while BALFAPPP showed high sensitivity but limited specificity when evaluating in-panel targets only. These factors should be considered when comparing results of cohorts with different sampling. BAL IL-1β showed potential in discriminating microbiologically confirmed VAP. Clinical Trial registration: NCT04766983, registered on February 23, 2021.

The prolonged incubation period of traditional culture methods leads to a delay in diagnosing invasive infections. Nanopore 16S rRNA gene sequencing (Nanopore 16S) offers a potential rapid diagnostic approach for directly identifying bacteria in infected body fluids. To evaluate the clinical utility of Nanopore 16S, we conducted a study involving the collection and sequencing of 128 monomicrobial samples, 65 polymicrobial samples, and 20 culturenegative body fluids. To minimize classification bias, taxonomic classification was performed using 3 analysis pipelines: Epi2me, Emu, and NanoCLUST. The result was compared to the culture references. The limit of detection of Nanopore 16S was also determined using simulated bacteremic blood samples. Among the three classifiers, Emu demonstrated the highest concordance with the culture results. It correctly identified the taxon of 125 (97.7%) of the 128 monomicrobial samples, compared to 109 (85.2%) for Epi2me and 102 (79.7%) for NanoCLUST. For the 230 cultured species in the 65 polymicrobial samples, Emu correctly identified 188 (81.7%) cultured species, compared to 174 (75.7%) for Epi2me and 125 (54.3%) for NanoCLUST. Through ROC analysis on the monomicrobial samples, we determined a threshold of relative abundance at 0.058 for distinguishing potential pathogens from background in Nanopore 16S. Applying this threshold resulted in the identification of 107 (83.6%), 117 (91.4%), and 114 (91.2%) correctly detected samples for Epi2me, Emu, and NanoCLUST, respectively, in the monomicrobial samples. Nanopore 16S coupled with Epi2me could provide preliminary results within 6 h. However, the ROC analysis of polymicrobial samples exhibited a random-like performance, making it difficult to establish a threshold. The overall limit of detection for Nanopore 16S was found to be about 90 CFU/ml. [ABSTRACT FROM AUTHOR]

Introduction: The aim of this study was to evaluate the performance of the Xpert MTB/RIF test in the diagnosis of M. tuberculosis in pulmonary and extrapulmonary clinical specimens with positive or negative smears. Materials and Methods: Between January 2018 and December 2021, a total of 2082 samples were examined, including 1526 respiratory samples and 556 non-respiratory samples. The samples processed for culture were inoculated into Löwenstein Jensen medium and Mycobacteria Growth Indicator Tube (MGIT) tubes, then MGITs were loaded into the MGIT 960 automated system. The Xpert MTB/RIF molecular test was performed to all samples according to the manufacturer’s recommendations. Results: M. tuberculosis was grown in culture in 153 (7.3%) of all samples, and the Xpert MTB/RIF test was positive in 203 (9.7%). ARB, MTB/RIF test and culture positivity in lung samples are; 86 (5.6%), 175 (11.4%) and 129 (8.4%), respectively. In extrapulmonary samples; the positivity was 7 (1.2%), 28 (5%) and 24 (4.3%). When mycobacterial culture results are accepted as reference, the sensitivity was 53.6% and the specificity was 99.4% for the Ehrlich-Ziehl-Neelsen staining method. For respiratory samples, these values were 58.1% and 99.2%; for extrapulmonary samples, sensitivity and specificity were 29.2% and 100%, respectively. For all the samples examined with The Xpert MTB/RIF test; sensitivity, specificity; positive predictive value; and negative predictive value were calculated as 89.5%, 96.6%, 67.5% and 99.1%, respectively. Conclusion: The sensitivity and specificity rates of the Xpert MTB/RIF test used in this study in non-respiratory samples were found to be slightly lower than in respiratory samples. It had high sensitivity and specificity rates in both sample groups. It was observed that The Xpert MTB/RIF test was a very fast and requiring low workload. [ABSTRACT FROM AUTHOR]

Background: The second wave of the coronavirus disease 2019 (COVID-19) pandemic recorded a surge in rhino-orbital-cerebral mucormycosis (ROCM) infection in COVID-19-positive patients with diabetes and on concomitant steroid therapy. The rapidly progressive and devastating nature of the disease necessitated prompt diagnosis and early intervention to improve patient outcomes. Histopathology and fungal culture remain essential tools; however, these investigations have long and variable turn-around times (TATs) and may delay the initiation of treatment. Frozen section is not widely available and should be avoided in COVID-19-positive cases due to the risk of aerosol production and droplet exposure. In cases with high clinicoradiologic suspicion for mucormycosis, imprint cytologic evaluation provides a rapid diagnosis. Familiarity with fungal cytomorphology, awareness of morphologic pitfalls, and implementation of a standardized reporting format aid in diagnostic accuracy. Method: Eighteen COVID-19-positive patients, who were admitted to our hospital with clinical suspicion of mucormycosis during June and July 2021, were included in the study. We used nasal or oral imprint cytology for the initial, rapid detection of Mucor. Cytology findings were correlated with histopathology and fungal culture results. Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were calculated. Results: The sensitivity, specificity, PPV, and NPV were 100%, 100%, 100% and 100%, respectively. Conclusion: This study showed that imprint cytology can be a rapid, cost-effective, first-line diagnostic modality in Mucor diagnosis. [ABSTRACT FROM AUTHOR]

Objective: To evaluate the association between the availability of GeneXpert®MTB/RIF in municipalities and the proportion of people who have access to this diagnostic technology for tuberculosis (TB), as well as the resistance detected by the surveillance system in Brazil. Methods: We analysed 4998 Brazilian municipalities that reported 432,937 new TB cases between 2015 and 2020. We compared municipalities with and without the availability of GeneXpert®MTB/RIF regarding the effective access to GeneXpert®MTB/RIF diagnosis and the prevalence of detected resistance. Results: Municipalities with at least one GeneXpert®MTB/RIF system had three times (95% CI 2.9–3.0) the access to diagnostic tests and 80.4% (95% CI 70.6%–90.2%) higher detection of resistance, compared with municipalities without this technology. We estimated that there have been 1890 cases of undetected resistance during this period in the country. Conclusions: The availability of GeneXpert®MTB/RIF system in the municipality increased the sensitivity of the surveillance for detecting TB resistance. Public Health Implications: It is a priority to strengthen laboratory networks and narrow the gap in access to rapid diagnosis in remote areas to improve the detection and control of drug‐resistant tuberculosis. [ABSTRACT FROM AUTHOR]

Rapid point-of-care tests that diagnose gonococcal infections and identify susceptibility to antibiotics enable individualized treatment. This could improve patient outcomes and slow the emergence and spread of antibiotic resistance. However, little is known about the long-term impact of such diagnostics on the burden of gonorrhea and the effective life span of antibiotics. We used a mathematical model of gonorrhea transmission among men who have sex with men in the United States to project the annual rate of reported gonorrhea cases and the effective life span of ceftriaxone, the recommended antibiotic for first-line treatment of gonorrhea, as well as 2 previously recommended antibiotics, ciprofloxacin and tetracycline, when a rapid drug susceptibility test that estimates susceptibility to ciprofloxacin and tetracycline is available. The use of a rapid drug susceptibility test with ≥50% sensitivity and ≥95% specificity, defined in terms of correct ascertainment of drug susceptibility and nonsusceptibility status, could increase the combined effective life span of ciprofloxacin, tetracycline, and ceftriaxone by at least 2 years over 25 years of simulation. If test specificity is imperfect, however, the increase in the effective life span of antibiotics is accompanied by an increase in the rate of reported gonorrhea cases even under perfect sensitivity. [ABSTRACT FROM AUTHOR]

Due to its high mortality rate, highly pathogenic avian influenza (HPAI), a notifiable animal illness designated by the World Organisation for Animal Health (WOAH), has caused enormous financial losses to the poultry sector. The H5 subtype of avian influenza virus (H5-AIV) is regarded as the most common highly pathogenic avian influenza virus (HPAIV) that threatens public health and safety. Virus isolation and reverse transcription quantitative PCR (RT-qPCR) are usually used to detect H5- AIV and are important for the timely diagnosis and control of H5-AIV. However, these methods are time-consuming and require a significant amount of effort. In this study, we established a recombinase-aided amplification (RAA) combined with CRISPR-Cas13a and lateral flow dipstick (LFD) assay for the detection of H5- AIV. The results showed that the process can be completed within 40 min at 37°C. The method had a detection limit of 0.1 copy/μL, which was comparable to the RT-qPCR. There was no cross-reactivity with H3-AIV, H7-AIV, H9-AIV, H10- AIV, IBV, NDV, RVA and DAstV. The kappa value of RT-RAA-Cas13a-LFD and RTqPCR in 380 clinical samples was 0.89 (κ>0.75). In conclusion, we established a convenient, efficient and accurate method to detect H5-AIV, and the results can be visualized and interpreted using LFD, which can be adapted to the needs of grassroots laboratories and field-deployable assays. This approach provides a new perspective for clinical H5-AIV diagnosis and has great potential for application in clinical quarantine of the poultry farming. [ABSTRACT FROM AUTHOR]

Objective：To improve the pre-processing laboratory test method for the severe acute respiratory syndome, coronavirus (SARS-CoV-2) specimens, and to evaluate its effectiveness. Methods：A total of 90 SARS-CoV-2 negative specimens and 30 SARS-CoV-2 positive specimens were collected. The specimens were divided into traditional group, modified 1 group, and modified 2 group, according to the test method. The specimens in traditional group were inactivated by using the guanidine salts and a 56 ℃, 30 minute water bath, followed by single tube oscillation, specimen de-identification, and single-handed lid opening for testing. The specimens in modified 1 group were inactivated by using the guanidine salts, followed by single tube oscillation, specimen de-identification, and single-handed lid opening for testing. The specimens in modified 2 group were inactivated by using the guanidine salts, followed by batch oscillation in vertical, horizontal, and 180-degree angle, specimen de-identification, and single-handed lid opening for testing. Real-time fluorescence quantitative PCR (RT-qPCR) method was used to detect the cycle threshold (Ct) values and detection time of the specimens in various groups； Spearman correlation analysis was used to analyze the correlation of Ct values between various groups； Blank-Altman method was used to perform the consistency test； the detection time of SARS-CoV-2 of the specimens in various groups was recorded. Results：The Ct values of the specimens in modified 1 group were strongly correlated with those in traditional group (P<0. 01), and the correlation coefficient(r)= 0. 975 4(0. 966 2－0. 982 1). The Ct values of the specimens in modified 2 group were strongly correlated with those in traditional group (P<0. 01), r=0. 986 2(0. 980 9－0. 990 0). The inter-group deviation of detecting SARS-CoV-2 negative specimens between modified 1 group and traditional group was 0. 158 2 (－0. 613 7－0. 930 1), and the inter-group deviation of detecting SARS-CoV-2 positive specimens was 0. 117 0(-0. 403 1－0. 637 1), indicating good consistency. The inter-group deviation between modified 2 group and traditional group for detecting SARS-CoV-2 negative specimens was 0. 015 7(-0. 550 4－ 0. 581 8), and for detecting SARS-CoV-2 positive specimens was 0. 022 7(-0. 454 1－0. 499 4), which indicated the good consistency. The detection time of 90 SARS-CoV-2 specimens in modified 1 group was 15 min, while the detection time of 90 SARS-CoV-2 specimens in modified 2 group was 10 min and the detection time of 90 SARS-CoV-2 specimens in traditional group was 45 min. Compared with traditional group, the detection time in modified 1 group was reduced by 67%and the detection time in modified 2 group was reduced by 11% . Conclusion：The modified SARS-CoV-2 specimen testing method shows strong correlation and good consistency with the traditional testing method. It can save the detection time and improve the efficiency. [ABSTRACT FROM AUTHOR]

RETRACTED ARTICLEThe aim of the study was to evaluate the relationship between chemotherapy and autoimmune reactions in patients with metastatic colorectal cancer. Cancer and autoimmunity are known to be interrelated, but until now it has been unclear to what extent chemotherapy specifically contributes to autoimmune reactions. We studied immunoglobulin M (IgM) levels in response to the administration of various human tissues before and during adjuvant chemotherapy. Patients received seven cycles of chemotherapy with the FOLFIRI plus cetuximab regimen. IgM levels against the tested tissues increased already after the first cycle of chemotherapy and continued to increase during the second and third cycles. Autoimmune responses then began to decrease from the fourth to seventh cycles, but remained elevated from baseline for most of the study tissues. Our results suggest that chemotherapy can induce a wide range of autoimmune reactions. Monitoring self-reactive IgM responses during treatment may help prevent or alleviate side effects associated with autoimmunity.

BackgroundRapid diagnostic tests (RDTs) for infectious diseases, with a turnaround time of less than 2 hours, are promising tools that could improve patient care, antimicrobial stewardship and infection prevention in the emergency department (ED) setting. Numerous RDTs have been developed, although not necessarily for the ED environment. Their successful implementation in the ED relies on their performance and impact on patient management.ObjectivesThe aim of this narrative review was to provide an overview of currently available RDTs for infectious diseases in the ED.SourcesPubMed was searched through August 2019 for available studies on RDTs for infectious diseases. Inclusion criteria included: commercial tests approved by the US Food and Drug Administration (FDA) or Conformité Européenne (CE) in vitro diagnostic devices with data on clinical samples, ability to run on fully automated systems and result delivery within 2 hours.ContentA nonexhaustive list of representative commercially available FDA- or CE-approved assays was categorized by clinical syndrome: pharyngitis and upper respiratory tract infection, lower respiratory tract infection, gastrointestinal infection, meningitis and encephalitis, fever in returning travellers and sexually transmitted infection, including HIV. The performance of tests was described on the basis of clinical validation studies. Further, their impact on clinical outcomes and anti-infective use was discussed with a focus on ED-based studies.ImplicationsClinicians should be familiar with the distinctive features of each RDT and individual performance characteristics for each target. Their integration into ED work flow should be preplanned considering local constraints of given settings. Additional clinical studies are needed to further evaluate their clinical effectiveness and cost-effectiveness.

Pathogenic microorganisms play a crucial role in the global disease burden due to their ability to cause various diseases and spread through multiple transmission routes. Immunity tests identify antigens related to these pathogens, thereby confirming past infections and monitoring the host’s immune response. Traditional pathogen detection methods, including enzyme-linked immunosorbent assays (ELISAs) and chemiluminescent immunoassays (CLIAs), are often labor-intensive, slow, and reliant on sophisticated equipment and skilled personnel, which can be limiting in resource-poor settings. In contrast, the development of microfluidic technologies presents a promising alternative, offering automation, miniaturization, and cost efficiency. These advanced methods are poised to replace traditional assays by streamlining processes and enabling rapid, high-throughput immunity testing for pathogens. This review highlights the latest advancements in microfluidic systems designed for rapid and high-throughput immunity testing, incorporating immunosensors, single molecule arrays (Simoas), a lateral flow assay (LFA), and smartphone integration. It focuses on key pathogenic microorganisms such as SARS-CoV-2, influenza, and the ZIKA virus (ZIKV). Additionally, the review discusses the challenges, commercialization prospects, and future directions to advance microfluidic systems for infectious disease detection.

Within the fields of infectious disease diagnostics, microfluidic-based integrated technology systems have become a vital technology in enhancing the rapidity, accuracy, and portability of pathogen detection. These systems synergize microfluidic techniques with advanced molecular biology methods, including reverse transcription polymerase chain reaction (RT-PCR), loop-mediated isothermal amplification (LAMP), and clustered regularly interspaced short palindromic repeats (CRISPR), have been successfully used to identify a diverse array of pathogens, including COVID-19, Ebola, Zika, and dengue fever. This review outlines the advances in pathogen detection, attributing them to the integration of microfluidic technology with traditional molecular biology methods and smartphone- and paper-based diagnostic assays. The cutting-edge diagnostic technologies are of critical importance for disease prevention and epidemic surveillance. Looking ahead, research is expected to focus on increasing detection sensitivity, streamlining testing processes, reducing costs, and enhancing the capability for remote data sharing. These improvements aim to achieve broader coverage and quicker response mechanisms, thereby constructing a more robust defense for global public health security.

Background: No univocal recommendation exists for microbiological diagnosis of ventilator-associated pneumonia (VAP). Sampling of either proximal or distal respiratory tract likely impacts on the broad range of VAP incidence between cohorts. Immune biomarkers to rule-in/rule-out VAP diagnosis, although promising, have not yet been validated. COVID-19-induced ARDS made VAP recognition even more challenging, often leading to overdiagnosis and overtreatment. We evaluated the impact of different respiratory samples and laboratory techniques on VAP incidence and microbiological findings in COVID-19 patients. Methods: Prospective single-centre cohort study conducted among COVID-19 mechanically ventilated patients in Policlinico Hospital (Milan, Italy) from January 2021 to May 2022. Microbiological confirmation of suspected VAP (sVAP) was based on concomitant endotracheal aspirates (ETA) and bronchoalveolar lavage (BAL). Conventional and fast microbiology (FILMARRAY® Pneumonia Panel plus, BALFAPPP) as well as immunological markers (immune cells and inflammatory cytokines) was analysed. Results: Seventy-nine patients were included. Exposure to antibiotics and steroid therapy before ICU admission occurred in 51/79 (64.6%) and 60/79 (65.9%) patients, respectively. Median duration of MV at VAP suspicion was 6 (5–9) days. Incidence rate of microbiologically confirmed VAP was 33.1 (95% CI 22.1–44.0) and 20.1 (95% CI 12.5–27.7) according to ETA and BAL, respectively. Concordance between ETA and BAL was observed in 35/49 (71.4%) cases, concordance between BALFAPPP and BAL in 39/49 (79.6%) cases. With BAL as reference standard, ETA showed 88.9% (95% CI 70.8–97.7) sensitivity and 50.0% (95% CI 28.2–71.8) specificity (Cohen's Kappa 0.40, 95% CI 0.16–0.65). BALFAPPP showed 95.0% (95% CI 75.1–99.9) sensitivity and 69% (95% CI 49.2–84.7) specificity (Cohen's Kappa 0.60, 95% CI 0.39–0.81). BAL IL-1β differed significantly between VAP (135 (IQR 11–450) pg/ml) and no-VAP (10 (IQR 2.9–105) pg/ml) patients (P = 0.03). Conclusions: In COVID-19 ICU patients, differences in microbial sampling at VAP suspicion could lead to high variability in VAP incidence and microbiological findings. Concordance between ETA and BAL was mainly limited by over 20% of ETA positive and BAL negative samples, while BALFAPPP showed high sensitivity but limited specificity when evaluating in-panel targets only. These factors should be considered when comparing results of cohorts with different sampling. BAL IL-1β showed potential in discriminating microbiologically confirmed VAP. Clinical Trial registration: NCT04766983, registered on February 23, 2021. [ABSTRACT FROM AUTHOR]

Acute bacterial meningitis (ABM) is a life threatening infection in children, associated with long term complications and high mortality rate.1,2 Gram staining and culture are routinely used for diagnosis of ABM. Antigen detection by latex agglutination can provide prompt results thereby facilitating early initiation of empirical antibiotic treatment. To estimate the proportion of Laboratory confirmed cases among children admitted with clinical suspicion of acute bacterial meningitis in a tertiary care hospital. To compare and analyse the diagnostic efficacy of Culture, Gram stain and antigen detection by Latex agglutination in Cerebrospinal fluid (CSF) samples for laboratory detection of Acute bacterial meningitis. CSF samples from pediatric patients with clinical suspicion of ABM were analysed by Gram stain, culture and Antigen detection by Latex agglutination method. Results were recorded and analysed. Of the 50 clinically suspected cases, 13(26%) were confirmed as Acute bacterial meningitis by laboratory investigations. Among the organisms identified, Streptococcus pneumoniae was the most common isolate in 5(38.46%) cases followed by Neisseria meningitidis, Klebsiella pneumoniae and Acinetobacter baumannii in 2(15.38%) cases each and Escherichia coli and Group B Streptococcus in 1(7.69%) case each. Among the confirmed cases, 7(53%) samples showed culture positivity while Gram stain identified 8(61.53%)cases. Latex agglutination test showed positivity in 9(69.23%) cases. In life threatening infections like acute bacterial meningitis, where early diagnosis and prompt treatment is of utmost importance, Latex agglutination test can provide results within minutes facilitating early initiation of empirical therapy, making it an effective adjunct to gram stain and culture. [ABSTRACT FROM AUTHOR]

Healthcare-associated pneumonia (HCAP) is a common nosocomial infection with high morbidity and mortality. Culture-based detection of the etiologic agent and drug susceptibility is time-consuming, potentially leading to the inadequate use of broad-spectrum empirical antibiotic regimens. The aim was to evaluate the diagnostic capabilities of rapid point-of-care multiplex polymerase chain reaction (PCR) assays from the endotracheal aspirate of critically ill patients with HCAP. A consecutive series of 29 intensive care unit (ICU) patients with HCAP and a control group of 28 patients undergoing elective surgical procedures were enrolled in the study. The results of the PCR assays were compared to the culture-based gold standard. The overall accuracy of the PCR assays was 95.12%, with a sensitivity of 92.31% and a specificity of 97.67%. The median time was 90 min for the rapid PCR tests (p < 0.001), while for the first preliminary results of the cultures, it was 48 h (46–72). The overall accuracy for rapid PCR testing in suggesting an adequate antibiotic adjustment was 82.98% (95% CI 69.19–92.35%), with a specificity of 90% (95% CI 55.50–99.75%), a positive predictive value of 96.77% (95% CI 83.30–99.92%), and a negative predictive value of 56.25 (95% CII 29.88–80.25%). This method of rapid point-of-care PCR could effectively guide antimicrobial stewardship in patients with healthcare-acquired pneumonia. [ABSTRACT FROM AUTHOR]