Rapid Diagnostic Method Research Articles

Pathological Mechanisms and Novel Testing Methods in Thrombotic Thrombocytopenic Purpura.

Thrombotic thrombocytopenic purpura (TTP) is an uncommon, but potentially disabling or even deadly, thrombotic microangiopathy with a well-studied mechanism of ADAMTS13 deficiency or dysfunction. While established treatments are largely effective, the standard ADAMTS13 testing required to definitively diagnose TTP may cause delays in diagnosis and treatment, highlighting the need for rapid and effective diagnostic methods. Additionally, the heterogeneous presentation and varied inciting events of TTP suggest more variation in its mechanism than previously thought, implying three potential pathways rather than the accepted two. The recent discovery of ADAMTS13 conformation as a potential contributor to TTP in addition to the proposal of using the absolute immature platelet count (A-IPC) as a biomarker, present novel areas for monitoring and treatment. A-IPC in particular may serve as a more rapid and accurate diagnostic test to distinguish TTP from non-TTP TMAs and to monitor treatment response and relapse. These considerations highlight the need to further study TTP in order to improve best practices and patient care.

Dual T-cell constant β chain (TRBC)1 and TRBC2 staining for the identification of T-cell neoplasms by flow cytometry

The diagnosis of leukemic T-cell malignancies is often challenging, due to overlapping features with reactive T-cells and limitations of currently available T-cell clonality assays. Recently developed therapeutic antibodies specific for the mutually exclusive T-cell receptor constant β chain (TRBC)1 and TRBC2 isoforms provide a unique opportunity to assess for TRBC-restriction as a surrogate of clonality in the flow cytometric analysis of T-cell neoplasms. To demonstrate the diagnostic utility of this approach, we studied 164 clinical specimens with (60) or without (104) T-cell neoplasia, in addition to 39 blood samples from healthy donors. Dual TRBC1 and TRBC2 expression was studied within a comprehensive T-cell panel, in a fashion similar to the routine evaluation of kappa and lambda immunoglobulin light chains for the detection of clonal B-cells. Polytypic TRBC expression was demonstrated on total, CD4+ and CD8+ T-cells from all healthy donors; and by intracellular staining on benign T-cell precursors. All neoplastic T-cells were TRBC-restricted, except for 8 cases (13%) lacking TRBC expression. T-cell clones of uncertain significance were identified in 17 samples without T-cell malignancy (13%) and accounted for smaller subsets than neoplastic clones (median: 4.7 vs. 69% of lymphocytes, p < 0.0001). Single staining for TRBC1 produced spurious TRBC1-dim subsets in 24 clinical specimens (15%), all of which resolved with dual TRBC1/2 staining. Assessment of TRBC restriction by flow cytometry provides a rapid diagnostic method to detect clonal T-cells, and to accurately determine the targetable TRBC isoform expressed by T-cell malignancies.

Development and Application of Droplet Digital PCR Assay for the Detection of Watermelon Silver Mottle Virus and Melon Yellow Spot Virus

Watermelon silver mottle virus (WSMoV) and melon yellow spot virus (MYSV) (Tospoviridae, Orthotospovirus) are responsible for silver mottle mosaic and yellow spot symptoms, posing threats to melon (Cucumis melo), watermelon (Citrullus lanatus), and cucumber and leading to significant economic losses in China. Early disease detection and monitoring of these two viruses are necessary for disease management, for which a rapid, reliable, and adaptable diagnostic method is required. In this study, using a droplet digital PCR (ddPCR) method, the conserved region of the nucleocapsid gene (N gene) sequence was detected in WSMoV and MYSV. The probes and primers for WSMoV and MYSV did not detect other relevant cucurbit viruses, and the specificity reached 100%. Although both qPCR and ddPCR exhibited good reproducibility, the reproducibility of ddPCR was better than that of qPCR. The reproducibility of ddPCR was proved to be 100%. Moreover, ddPCR exhibited a good linear correlation with varying concentrations of targets. The detection limits of WSMoV and MYSV in ddPCR were 18 and 9 copies/μL and were approximately 12- and 18-times more than those in qPCR, respectively. Finally, 62 samples collected from the field (including infected melon, watermelon, and weeds) were further evaluated for the presence of WSMoV and MYSV. The field samples exhibited 91.94% and 51.61% positivity rates in ddPCR assays for WSMoV and MYSV, respectively; the rates were higher than those in qPCR (59.68% and 43.39%, respectively). The results indicated that ddPCR has a higher accuracy than qPCR. Therefore, ddPCR could be used in the clinical diagnosis of early infections of WSMoV and MYSV. To the best of our knowledge, this is the first study to establish a ddPCR method for the detection of WSMoV and MYSV. The application of this method for differential detection of MYSV and WSMoV will help in understanding the epidemics caused by these two important viruses and provide important information for the early detection, monitoring, and rapid extermination of infection.

The development of RT-RPA and CRISPR-Cas12a based assay for sensitive detection of infectious hematopoietic necrosis virus (IHNV)

Infectious hematopoietic necrosis virus (IHNV) is an economically important virus causing significant mortalities among wild and cultured salmonid fish worldwide. Rapid and sensitive diagnostic methods of IHNV are crucial for timely controlling infections. For better detection of IHNV, we have established a detection technology based on the reverse transcription and recombinase polymerase amplification (RT-RPA) and CRISPR/Cas12a to detect the N gene of IHNV in two steps. Following the screening of primer pairs, the reaction temperature and time for RPA were optimized to be 41 °C and 35 min, respectively, and the CRISPR/Cas12a reaction was performed at 37 °C for 15 min. The whole detection procedure including can be accomplished within one hour, with a detection sensitivity of about 9.5 copies/µL. The detection method exhibited high specificity with no cross-reaction to the other Novirhabdoviruses HIRRV and VHSV, allowing naked-eye interpretation of the results through lateral flow or fluorescence under ultraviolet light. Overall, our results demonstrated that the developed RT-RPA-Cas12a-mediated assay is a rapid, specific and sensitive detection method for routine and on-site detection of IHNV, which shows a great application promise for the prevention of IHNV infections.

Phospho-Tau 181 quantification method for Alzheimer's disease based on an array 2D biosensor combined with surface plasmon resonance imaging

Alzheimer's disease is among the neurodegenerative diseases for which there is a lack of rapid, effective, and non-invasive diagnostic methods. The development of a phospho-Tau 181 assay biosensor is therefore a response to the need for methods to diagnose AD. The present work was aimed at developing a fast, selective, and repeatable method for the quantitative determination of phospho-Tau 181, which could be used even during routine blood tests. Our method is a form of what is called liquid biopsy. The developed method underwent validation, as a result of which its analytical parameters were determined.An LOQ of 3.35 pg mL−1 was obtained, confirming the possibility of trace analysis of phospho-Tau 181 in human plasma. Relative percentage error values below 15 % and CVs in the range 1.47–7.09 % attest to the high accuracy and precision of the presented method. Also, the sample matrix was not found to significantly affect the results obtained for phospho-Tau 181 concentrations. The new SPRi biosensor provides reproducible measurements of the analyte under study (CV = 3.18–4.26 %). Although the method requires absolute adherence to the recommendations of the analytical procedure protocol, it achieves high selectivity and provides 90 % certainty of the correctness of the diagnosis based on measurements of phospho-Tau 181 concentration.

Assessing heterogeneity of patient and health system delay among TB in a population with internal migrants in China.

The diagnostic delay of tuberculosis (TB) contributes to further transmission and impedes the implementation of the End TB Strategy. Therefore, we aimed to describe the characteristics of patient delay, health system delay, and total delay among TB patients in Shanghai, identify areas at high risk for delay, and explore the potential factors of long delay at individual and spatial levels. The study included TB patients among migrants and residents in Shanghai between January 2010 and December 2018. Patient and health system delays exceeding 14 days and total delays exceeding 28 days were defined as long delays. Time trends of long delays were evaluated by Joinpoint regression. Multivariable logistic regression analysis was employed to analyze influencing factors of long delays. Spatial analysis of delays was conducted using ArcGIS, and the hierarchical Bayesian spatial model was utilized to explore associated spatial factors. Overall, 61,050 TB patients were notified during the study period. Median patient, health system, and total delays were 12 days (IQR: 3-26), 9 days (IQR: 4-18), and 27 days (IQR: 15-43), respectively. Migrants, females, older adults, symptomatic visits to TB-designated facilities, and pathogen-positive were associated with longer patient delays, while pathogen-negative, active case findings and symptomatic visits to non-TB-designated facilities were associated with long health system delays (LHD). Spatial analysis revealed Chongming Island was a hotspot for patient delay, while western areas of Shanghai, with a high proportion of internal migrants and industrial parks, were at high risk for LHD. The application of rapid molecular diagnostic methods was associated with reduced health system delays. Despite a relatively shorter diagnostic delay of TB than in the other regions in China, there was vital social-demographic and spatial heterogeneity in the occurrence of long delays in Shanghai. While the active case finding and rapid molecular diagnosis reduced the delay, novel targeted interventions are still required to address the challenges of TB diagnosis among both migrants and residents in this urban setting.

Modeling Anisotropic Electrical Conductivity of Blood: Translating Microscale Effects of Red Blood Cell Motion into a Macroscale Property of Blood.

Cardiovascular diseases are a leading global cause of mortality. The current standard diagnostic methods, such as imaging and invasive procedures, are relatively expensive and partly connected with risks to the patient. Bioimpedance measurements hold the promise to offer rapid, safe, and low-cost alternative diagnostic methods. In the realm of cardiovascular diseases, bioimpedance methods rely on the changing electrical conductivity of blood, which depends on the local hemodynamics. However, the exact dependence of blood conductivity on the hemodynamic parameters is not yet fully understood, and the existing models for this dependence are limited to rather academic flow fields in straight pipes or channels. In this work, we suggest two closely connected anisotropic electrical conductivity models for blood in general three-dimensional flows, which consider the orientation and alignment of red blood cells (RBCs) in shear flows. In shear flows, RBCs adopt preferred orientations through a rotation of their membrane known as tank-treading motion. The two models are built on two different assumptions as to which hemodynamic characteristic determines the preferred orientation. The models are evaluated in two example simulations of blood flow. In a straight rigid vessel, the models coincide and are in accordance with experimental observations. In a simplified aorta geometry, the models yield different results. These differences are analyzed quantitatively, but a validation of the models with experiments is yet outstanding.

Fluorescent polymer as a biosensing tool for the diagnosis of microbial pathogens

Diseases and diagnoses are predominant in the human population. Early diagnosis of etiological agents plays a vital role in the treatment of bacterial infections. Existing standard diagnostic platforms are laborious, time-consuming, and require trained personnel and cost-effective procedure, though they are producing promising results. These shortcomings have led to a thirst for rapid diagnostic procedures. Fluorescence-based diagnosis is one of the efficient rapid diagnostic methods that rely on specific and sensitive bacterial detection. Emerging bio-sensing studies on conducting polymers (CPs) are gaining popularity in medical diagnostics due to their promising properties of high fluorescence efficiency, good light stability, and low cytotoxicity. Poly[2-methoxy-5-(2′-ethylhexyloxy)-1,4-phenylenevinylene] (MEH-PPV), is the first identified soluble polymer and model material for understanding the fundamental photophysics of conventional CPs. In this present study, MEH-PPV is used as a fluorescent dye for direct pathogen detection applications by interacting with the microbial cell surface. An optimized concentration of MEH-PPV solution used to confirm the presence of selective bacterial structures. The present study endeavours towards bacterial detection based on the emission from bacteria due to interfacial interaction between polymer and bacterial surface.

Preparation of Monoclonal Antibodies against the Capsid Protein and Development of an Epitope-Blocking Enzyme-Linked Immunosorbent Assay for Detection of the Antibody against Porcine Circovirus 3.

Porcine circovirus type 3 (PCV3) is endemic in swine worldwide and causes reproductive disorders, dermatitis and nephrotic syndrome, and multi-organ inflammation. Currently, there is a growing need for rapid and accurate diagnostic methods in disease monitoring. In this study, four monoclonal antibodies (mAbs) against PCV3 capsid proteins were prepared (mAbs 2F6, 2G8, 6E2, and 7E3). MAb 7E3, which had the highest binding affinity for the Cap protein, was chosen for further investigation. A novel B cell epitope 110DLDGAW115 was identified using mAb 7E3. An epitope-blocking (EB) enzyme-linked immunosorbent assay (ELISA) was successfully developed using horseradish-peroxidase-labeled mAb 7E3 to detect PCV3 antibodies in porcine sera. Moreover, the EB-ELISA showed no specific reaction with other porcine disease sera, and the cut-off value was defined as 35%. Compared with the commercial ELISA, the percentage agreement was 95.59%. Overall, we have developed a novel EB-ELISA method that accurately and conveniently detects PCV3 in serum, making it a valuable tool for the clinical detection of PCV3 infection.

Rapid Detection of the Strawberry Foliar Nematode Aphelenchoides fragariae Using Recombinase Polymerase Amplification Assay with Lateral Flow Dipsticks.

Rapid and reliable diagnostic methods for plant-parasitic nematodes are critical for facilitating the selection of effective control measures. A diagnostic recombinase polymerase amplification (RPA) assay for Aphelenchoides fragariae using a TwistAmp® Basic Kit (TwistDx, Cambridge, UK) and AmplifyRP® Acceler8® Discovery Kit (Agdia, Elkhart, IN, USA) combined with lateral flow dipsticks (LF) has been developed. In this study, a LF-RPA assay was designed that targets the ITS rRNA gene of A. fragariae. This assay enables the specific detection of A. fragariae from crude nematode extracts without a DNA extraction step, and from DNA extracts of plant tissues infected with this nematode species. The LF-RPA assay showed reliable detection within 18-25 min with a sensitivity of 0.03 nematode per reaction tube for crude nematode extracts or 0.3 nematode per reaction tube using plant DNA extracts from 0.1 g of fresh leaves. The LF-RPA assay was developed and validated with a wide range of nematode and plant samples. Aphelenchoides fragariae was identified from seed samples in California. The LF-RPA assay has great potential for nematode diagnostics in the laboratory with minimal available equipment.

Diagnostic Tool for the Identification of Bactrocera dorsalis (Hendel) (Diptera: Tephritidae) Using Real-Time PCR.

Accurate identification of Bactrocera dorsalis (Hendel) (Diptera: Tephritidae), commonly known as the Oriental fruit fly, is a significant challenge due to the morphological convergence and taxonomic uncertainties of species belonging to the same genus. This highly polyphagous species poses a significant threat to fruit crops. With its potential establishment in Europe becoming a growing concern, there is an urgent need for rapid and efficient diagnostic methods. The study presented here introduces a diagnostic protocol based on real-time PCR using a TaqMan probe for the early and reproducible identification of B. dorsalis. Specimens representing the genetic diversity of the Italian population were collected and analyzed. Specific primers and probe were designed based on the conserved regions and an in silico analysis confirmed their specificity. The assay conditions were optimized, and analytical sensitivity, specificity, repeatability, and reproducibility were evaluated. The protocol showed high sensitivity and specificity, accurately detecting low DNA concentrations of B. dorsalis. This standardized method provides a reliable tool for routine diagnostics, enhancing the accuracy and efficiency of identifying the Oriental fruit fly at all stages of its development, thereby facilitating effective pest management measures. The development of this diagnostic protocol is crucial for monitoring and supporting efforts to prevent the passive spread of B. dorsalis in Europe, particularly in light of the recent active infestations detected in Italy.

Innovative laboratory methods for improved tuberculosis diagnosis and drug-susceptibility testing

The laboratory plays a vital role in the diagnosis of all clinical forms of tuberculosis (TB), from microbiological confirmation of Mycobacterium tuberculosis to drug-susceptibility testing (DST) and treatment monitoring. For many decades, laboratory diagnosis of tuberculosis was based on conventional methods such as smear microscopy, and culture-based methods. However, Mycobacterium tuberculosis is a slow-growing organism, requiring 2–4 weeks or longer for cultures to yield results. Therefore, the evaluation of novel and rapid diagnostic methods has been a priority for research and development. In the beginning of 1990s, molecular-based diagnostics became widely available providing rapid detection, identification, and DST of M. tuberculosis. In this paper, we review some of the new diagnostic methods introduced in the clinical laboratory for the diagnosis of tuberculosis. With the global goal of ending TB as a public health challenge by 2030, enhancing diagnostic capabilities for latent and active TB, along with improving DST, would improve identification and management of cases, reducing transmission rates and curbing the spread of drug-resistant strains. These innovations promise to transform TB control efforts, bringing us closer to eradicating this persistent global health threat.

Нарушения опорно-двигательного аппарата у водолазного состава военно-морского флота

Aim. To study the features of the pathogenesis and clinical picture of musculoskeletal disorders in various categories of military personnel of the Navy (Navy) and pensioners of the Ministry of Defense of the Russian Federation (MOD) based on a set of clinical, instrumental and laboratory data. Design. Prospective observational study. Materials and methods. An examination was carried out and the medical documentation was studied of 98 military personnel of the Navy and pensioners of the Defense Ministry in the following groups: I — military personnel of the Navy without a history of exposure to hyperbaria, II — military personnel belonging to deep-sea divers with moderate work experience, III — military personnel belonging to deep-sea divers with significant work experience, IV — pensioners of the Ministry of Defense, whose professional activity was related to work on deep-sea vehicles (aquanauts), V — pensioners of the Ministry of Defense without a history of exposure to hyperbaria. The patients underwent a detailed laboratory and instrumental study of bone tissue metabolism, including digital radiography, osteodensitometry, magnetic resonance imaging (MRI) and computed tomography. Results. Significant osteo-articular changes of the type of dysbaric osteonecrosis were noted among Navy diving personnel, most pronounced among retired Navy personnel – aquanauts. Conclusion. Osteoarticular changes in Navy diving personnel are of a specific dysbarogenic nature, polyetiological, localized in large joints and diaphyses of adjacent bones and become significant as the length of service, the severity of the work performed and occupational risks in hyperbaric conditions increase. Digital radiography and MRI are recognized as the optimal methods for diagnosing dysbaric osteonecrosis and “dysbarogenic osteoarthropathy”. As a rapid diagnostic method, it is advisable to use innovative urine test strips that allow determining the threshold concentrations of bone resorption markers — β-crosslaps and α-crosslaps. Keywords: diving personnel of the Navy, aquanauts, dysbaric osteonecrosis and "dysbarogenic osteoarthropathy", etiology, pathogenesis, clinical picture, diagnosis.

Antibody Detection of Toxoplasma gondii on Blood Donor at PMI Kota Yogyakarta by Rapid Diagnostic Methods in 2022

Toxoplasmosis is a disease that caused by Toxoplasma gondii parasite. Toxoplasma gondii is a group of protozoa, and it belongs to intracellular obligate parasites. This parasite mostly found in animal, and it can be dangerous, on many cases, it can cause death with no symptoms. The purpose of this study is to find out the percentage of IgG and IgM antibodies examination of Toxoplasma gondii on blood donor in PMI Kota Yogyakarta. This study belongs to descriptive observational type, with cross-sectional design to find out the results of antibody detection on Toxoplasma gondii. The detection was carried out by Rapid Diagnostic Methods that can detect the presence of IgG and IgM antibodies. This study was also using questionnaires to know the relationship between the daily behaviours with toxoplasmosis case. The results show that 14 samples were reactive on IgG antibody parameter (76%) and 11 samples were non-reactive (24%). From the questionnaires known that the daily behaviour’s such as having a cat, eating raw/undercooked meat products, eating unwashed fruits and vegetables related to the case of toxoplasmosis.

Detection of Toxoplasma gondii Parasite on Blood Donor at PMI Kabupaten Magelang by Rapid Diagnostic Methods in 2022

Toxoplasma gondii is a parasite that can infect most warm-blooded animals, including humans. The infection is caused by Toxoplasma gondii called Toxoplasmosis. Toxoplasmosis transmission can occur because of human behaviour’s such as keeping cats, eating raw vegetables and fruit with a less clean, not washing hands properly, and consuming food and drinks served without lids that could potentially be contaminated with oocysts, as well as soil-related work such as gardening and farming. Toxoplasma gondii is an infectious parasite that could be transmitted through blood transfusions. The aim of this research was to detect IgM and IgG antibodies of Toxoplasma gondii in blood donors by Rapid Diagnostic method at PMI Kabupaten Magelang. This research is a descriptive observational with a cross sectional design and used 25 respondents with blood categories A, B, O, and AB at PMI Kabupaten Magelang in 2022. According to the results of the Toxoplasmosis examination utilizing the Rapid Diagnostic Test technique on blood donors at PMI Kabupaten Magelang, as many as 25 respondents obtained non-reactive IgM and IgG antibodies against Toxoplasma gondii. It could be concluded that no respondents were found to have reactive IgM and IgG antibodies against Toxoplasma gondii.

A Retrospective Analysis of Diagnostic Accuracy of Frozen Sections Compared to Permanent Sections: A 5 Years Study in A Single Tertiary Care Hospital In South Delhi

Introduction: Intraoperative frozen section pathology consultation has long been applied to verify/categorize a lesion, evaluate surgical margins, determine the organ of origin, and assess tissue adequacy for further diagnostic studies. Correlation of intraoperative frozen section diagnosis with final diagnosis can be an important component of an institution’s quality assurance process. The aim of this study was to evaluate the concordance and discordance between frozen section and histopathology diagnosis and to assess the reliability and challenges associated with this rapid diagnostic method. Materials and Methods: A retrospective review of all intraoperative consultation in the Frozen Section and Histopathology Department of Pathology, Hamdard Institute of Medical Sciences and Research and Hospital, New Delhi from 1st January 2018 to 31st December 2022 was performed. During this five-year study period, a total of 185 patients from surgical departments underwent intraoperative consultation. The final diagnosis from the surgical pathology report was compared to the intraoperative consultation diagnosis. Results: Frozen section diagnoses were evaluated on all the 185 cases, with 88 cases identified as positive for malignancy and 97 cases as negative for malignancy. On subsequent histopathology paraffin sections, 92 cases were positive and 93 negative for malignancy. The comparison revealed an approximate result concordance of 97.0% and a discordance of 3.0%. Conclusion: Frozen section intraoperative consultation is a reliable rapid method for patient diagnosis and management. Monitoring diagnostic accuracy of intraoperative consultation provides substantial information towards the causes of the errors. Our study suggests that more accurate sampling, and knowledge about clinical history and presentation can reduce the limitation and increase the diagnostic accuracy thus avoiding technical errors and reducing result discordance.

A novel Leifsonia xyli subsp. xyli quantitative LAMP-based diagnostic correlated with sugarcane ratoon stunting disease rating

Context Ratoon stunting disease (RSD), caused by Leifsonia xyli subsp. xyli (Lxx), poses a significant economic threat to sugarcane (Saccharum hybrid) worldwide. RSD is hard to manage due to its elusive visible symptomology and disease rating of cultivars is subjective. Aims We aimed to develop a sensitive, rapid, and quantitative Lxx diagnostic method able to correlate Lxx titre and disease resistance rating of sugarcane cultivars. Methods A Lxx diagnostic method was developed using heat lysis-based reagent-free DNA isolation from xylem sap followed by loop-mediated isothermal amplification (LAMP)-based colorimetric and fluorescence quantification within a single microcentrifuge tube. Bacterial titre was then statistically correlated with industry-agreed disease resistance ratings for key sugarcane cultivars. Key results The diagnostic was highly sensitive (1 cell/μL) and reproducible (%s.d. = &lt;5%, for n = 3), and showed excellent linear dynamic range (i.e. 10 pM−1 aM or 107−100 copies/μL, r = 0.99) for quantitative Lxx detection. LAMP quantifications were completely concordant with quantitative polymerase chain reaction quantification from the same samples. Additionally, a strong correlation was determined between the detected quantitative bacterial titres and known cultivar disease resistance ratings (r = 0.82, n = 10, P &lt; 0.001). Conclusion The novel LAMP-based Lxx diagnostic was validated as a fast, simple, and relatively cost-effective means of RSD resistance rating, making it a reliable contribution towards RSD management. Implications The development of this diagnostic tool provides a practical solution for accurately measuring Lxx titre and assessing disease resistance in sugarcane plants, aiding in effective risk management of RSD spread, and mitigating its economic impact on sugarcane crops worldwide.

Development of alternative diagnosis of HH1, HH3, HH5 and HCD fertility haplotypes and subfertility syndrome in cattle

AbstractThe increasing prevalence of hereditary anomalies in Holstein cattle populations presents a pressing issue, leading to concerns such as embryonic mortality and the birth of non‐viable offspring. This study addresses the urgency of managing harmful genetic mutations in Holstein cattle by developing alternative diagnostic methods. The research aims to devise effective means to diagnose fertility haplotypes HH1, HH3, HH5, HCD and BY and subfertility syndrome in cattle. To achieve this goal, a range of molecular genetic techniques were employed, including Tetra‐Primer ARMS‐PCR methods, PCR‐RFLP analysis and allele‐specific PCR. These methods facilitated the identification of heterozygous carriers of various fertility haplotypes and subfertility syndrome in Holstein cows and servicing bulls. The study reveals the prevalence of these genetic defects within the Republic of Kazakhstan's cattle population. HH1, HH3, HH5, HCD and BY fertility haplotypes were found to have occurrence rates ranging from 1.4% to 16.6%, with subfertility syndrome detected in 4.5% of Simmental bulls. The practical significance of this research lies in its contribution to genetic monitoring and management strategies for Holstein cattle populations. By introducing affordable, rapid and accurate diagnostic methods, such as the T‐ARMS‐PCR, the study provides a valuable tool for controlling and mitigating the spread of harmful genetic mutations, ultimately improving the overall genetic health and productivity of Holstein cattle in the region. This research addresses a critical need in the cattle breeding industry and underscores the importance of genetic monitoring to ensure the long‐term viability and sustainability of Holstein cattle populations.

The Challenges of The Diagnostic and Therapeutic Approach of Patients with Infectious Pathology in Emergency Medicine.

A retrospective observational study was performed on 657 patients, from whom clinical samples (urine, purulent secretions, blood cultures, etc.) were collected for microbiological diagnosis in the first 3 days after presentation in the ED. The identification of pathogens and the antimicrobial susceptibility testing with minimum inhibitory concentration determination were carried out according to the laboratory protocols. From the 767 biological samples analyzed, 903 microbial isolates were identified. E. coli was most frequently isolated (24.25%), followed by Klebsiella spp., S. aureus (SA), and non-fermentative Gram-negative bacilli. E. coli strains maintained their natural susceptibility to most antibiotics tested. In the case of Pseudomonas spp. and Acinetobacter spp., increased rates of AMR were identified. Also, 32.3% of SA strains were community-acquired MRSA. The introduction of rapid microbiological diagnostic methods in emergency medicine is imperative in order to timely identify AMR strains and improve therapeutic protocols.

Scoping review of risk-scoring tools for early prediction of bloodstream infections caused by carbapenem-resistant Enterobacterales: do we really have a reliable risk-scoring tool?

Bloodstream infections (BSIs) caused by carbapenem-resistant Enterobacterales (CRE) are a global health concern. Rapid identification of CRE may improve patient outcomes and reduce inappropriate antibiotic prescription. The use of risk-scoring tools (RSTs) can be valuable for optimizing the decision-making process for empirical antibiotic therapy of suspected CRE bacteraemia. These tools can also be used to triage use of expensive rapid diagnostic methods. We systematically reviewed the relevant literature in PubMed/MEDLINE, CINAHL, Cochrane, Web of Science, Embase and Scopus up to 1 November 2022 to identify RSTs that predict CRE BSIs. The literature review and analysis of the articles were performed by two researchers; any inconsistencies were resolved through discussion. We identified 9 RSTs developed for early prediction of CRE BSIs and only logistic regression was used for most studies. These RSTs were quite different from each other in terms of their performance and the variables they included. They also had notable limitations and very few of them were externally validated. RSTs for early prediction of CRE BSIs have limitations and lack of external validity outside the local setting in which they were developed. Future studies to identify optimal RSTs in high and low CRE-endemic settings are warranted. Approaches based on rapid diagnostics and RSTs should be compared with a treatment approach using both methods in a randomized controlled trial.