Search Results (388)

Background: Accurate differentiation between Mycobacterium tuberculosis (MTB) and nontuberculous mycobacteria (NTM) is essential for proper diagnosis and treatment. This study compares the diagnostic performance of two commercial real-time PCR kits, AdvanSure^TM TB/NTM and Kogene PowerChek^TM MTB/NTM, for detecting MTB, NTM, and negative (no growth, NG) clinical specimens. Methods: A total of 390 clinical residual specimens were collected from patients between December 2022 and June 2023. The samples, including sputum, bronchoalveolar lavage, tracheal aspirate and body fluid, were initially tested with MGIT culture and then analyzed using both PCR kits. Sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and overall accuracy were evaluated. Discrepant results between the two PCR assays were further investigated using sequencing to identify the detected mycobacterial species, and final diagnoses were verified by culture results and review of electronic medical records. Results: Of the 390 specimens, both AdvanSure^TM and PowerChek^TM real-time PCR assays demonstrated 100% sensitivity for both MTB and NTM detection. For MTB detection, AdvanSure^TM demonstrated a specificity of 100%, with a PPV, NPV, and overall accuracy all reaching 100%. In comparison, PowerChek^TM showed a specificity of 98.62%, a PPV of 96.15%, an NPV of 100%, and an overall accuracy of 98.97%. For NTM detection, both AdvanSure^TM and PowerChek^TM exhibited identical performance metrics. The specificity was 99.58% for both assays, with a PPV of 99.34%, NPV of 100%, and an overall accuracy of 99.74%. Five discrepant results were finally confirmed as four NTM detection cases and one negative case by culture and clinical diagnosis which showed four cases of PowerChek^TM MTB+NTM detection and one case of NTM detection, respectively. Conclusions: The PowerChek^TM MTB/NTM real-time PCR kit demonstrated excellent diagnostic performance for the detection of MTB and NTM, with high sensitivity, specificity, and accuracy. Minor discrepancies, particularly in detecting MTB+NTM mixed infections, highlight the importance of complementary sequencing analysis for resolving uncertain results. These findings support the clinical utility of both PCR assays as reliable tools for rapid diagnosis of mycobacterial infections. PowerChek^TM showed occasional false positives, suggesting that optimizing the assay’s cutoff threshold or amplification parameters could enhance its specificity and reduce false-positive results in clinically ambiguous cases. Full article

(1) Background: Hemorrhagic fever with renal syndrome (HFRS) remains a prevalent zoonosis in Eurasia. Orthohantavirus puumalaense (PUUV), carried by bank voles (Myodes glareolus), is the principal zoonotic pathogen of HFRS in this region. Despite ongoing efforts to develop effective drugs and vaccines against PUUV, this challenge remains. (2) Aim: In this study, we aimed to express a large quantity of the PUUV recombinant N (rN) protein using E. coli. We also sought to develop a protocol for extracting the rN protein from pellets, solubilizing, and refolding it to restore its native form. This protocol is crucial for producing a large quantity of rN protein to develop vaccines and diagnostic tools for HFRS. (3) Methods; PUUV S segment open reading frame (ORF) coding for N protein was synthesized and cloned into the plasmid vector pET-28 (A+). The ORF was transformed, expressed and induced in BL21(DE3) pLysS E. coli strain. Subsequently, rN protein was purified using immobilized metal affinity and ion chromatography. Immune reactivity of rN protein was tested by employing in house and commercial VektoHanta-IgG kit ELISA methods (both in vitro and in vivo). (4) Results: The best conditions for scaling up the expression of the PUUV rN protein were an incubation temperature of 20 °C during a 20 h incubation period, followed by induction with 0.5 mM IPTG. The most significant protein yield was achieved when the pellets were incubated in denaturing buffer with 8M urea. The highest yield of refolded proteins was attained using non-denaturing buffer (50 mM Tris-HCl) supplemented with arginine. A final 50 μL of PUUV rN protein solution with a concentration of 7 mg/mL was recovered from 1 L of culture. The rN protein elicited an antibody response in vivo and reacted with serum taken from patients with HFRS by ELISA in vitro. (5) Conclusion: Therefore, the orthohantavirus N protein’s ability to elicit immune response in vivo suggests that it can be used to develop vaccines against PUUV after conducting in vitro and in vivo studies to ascertain neutralising antibodies. Full article

Background/Objectives: Neutralizing autoantibodies against type I interferons, particularly interferon-alpha (IFN-α), have been implicated in severe COVID-19 outcomes. This study investigated the prevalence and functional significance of anti-IFN-α autoantibodies (AAbs) in hospitalized unvaccinated COVID-19 patients and their association with COVID-19 disease severity. Methods: We retrospectively analyzed serum samples from 122 hospitalized COVID-19 patients (asymptomatic/mild: n = 69, moderate: n = 35, severe/critical: n = 18) and 32 healthy uninfected controls. Anti-IFN-α AAbs were quantified using a commercial enzyme-linked immunosorbent assay (ELISA) kit, with functional neutralization assessed via competitive ELISA and STAT1 phosphorylation inhibition. Statistical comparisons were performed using one-way ANOVA for parametric data and the Kruskal–Wallis test for non-parametric variables. Results: Anti-IFN-α AAbs were detected in 24.6% of COVID-19 patients, with all clinical subgroups showing significantly higher titers compared to healthy controls (p < 0.05). Although no significant differences in anti-IFN-α AAb levels were found between mild, moderate, and severe cases, patients with severe or critical COVID-19 had markedly higher mean titers (10,511.3 ng/mL) compared to non-severe (mild + moderate) cases (375.2 ng/mL, p < 0.001). Strongly neutralizing anti-IFN-α AAbs, with high titers (>20,000 ng/mL) and the ability to inhibit STAT1 phosphorylation, were identified in three severe COVID-19 cases. Anti-IFN-α AAb levels correlated positively with CRP (r = 0.80, p < 0.0001), LDH (r = 0.80, p = 0.001), and neutrophil count (r = 0.52, p = 0.003), and negatively with lymphocyte count (r = −0.59, p = 0.0006). Conclusions: Elevated and functionally neutralizing anti-IFN-α AAbs were associated with severe COVID-19. These findings support their role as a risk factor for poor outcomes and emphasize the importance of early COVID-19 vaccination. Screening may help identify high-risk individuals, particularly those unvaccinated or with immune vulnerabilities. Full article

Bovine leukemia virus (BLV) remains a major concern for cattle industries worldwide due to its persistent nature, economic impact, and challenges in control. In this study, we conducted a comprehensive nationwide survey of BLV in Kazakhstan between 2014 and 2024, utilizing serological diagnostics to assess prevalence and characterize viral genotypes (2024). A total of 433,537 serum samples were screened by agar gel immunodiffusion (AGID), revealing an overall seroprevalence of 5.87%, with the highest rates observed in the North Kazakhstan, Kostanay, and East Kazakhstan regions. In 2024, a targeted analysis of 3736 serum and 536 whole blood samples across 17 regions was performed using AGID, ELISA, real-time PCR, and nested PCR. ELISA demonstrated higher sensitivity than AGID (10.4% vs. 8.2%), confirmed by statistical correlation (r = 0.97, p < 0.001) and a Wilcoxon signed-rank test (p = 0.026). Real-time PCR detected BLV DNA in 4.7% of samples, with the highest positivity in the East Kazakhstan and Abai regions, confirming active viral circulation. Validation of a domestically developed AGID diagnostic kit showed full concordance with commercial assays (IDEXX, IDvet), supporting its use in national surveillance programs. These findings highlight the endemic status of BLV in Kazakhstan. Molecular analysis of sequenced isolates revealed the presence of genotype G-7, consistent with strains circulating in neighboring countries. Together, these results underscore the importance of integrated serological and molecular approaches for effective monitoring and control. Full article

Circulating cell-free nucleic acids (NAs), in particular plasma-derived cell-free DNA, have evolved into promising clinical analytes for prenatal diagnostics, cancer analysis, and cancer surveillance and therapy monitoring. Nevertheless, salivary extracellular and extracellular vesicle (EV)-derived DNA and microRNA have recently gained attention as potential non-invasive biomarkers for a variety of diseases, including cancer, cardiovascular, autoimmune, and infectious diseases. Our goal in this study was therefore to evaluate and optimize commercially available approaches for cell-free nucleic acid isolation, focusing specifically on DNA and miRNA present in cell-free saliva or saliva-derived EVs. Along these lines, we investigated various commercially available kits, which enable parallel isolation of cell-free DNA and RNA in separate fractions from cell-free saliva and salivary EVs, respectively, and compared them to single analyte extraction kits. The efficiency of all tested nucleic acid extraction methods was determined by comparing DNA and RNA fluorescence spectroscopy measurements and quantitative PCR values obtained from a selection of different DNA- and microRNA targets. We found the Norgen Plasma/Serum RNA/DNA Purification Mini kit in combination with the miRCURY exosome isolation kit to work best in our hands and to provide the highest yields of EV-derived nucleic acids. Having tested and identified effective protocols for isolating salivary extracellular nucleic acids, we present with this comparison study, among others, a sound basis for future circulating small nucleic acid and epigenetic biomarker research aiming for early disease diagnosis, prognosis, and prediction from cell-free saliva, representing an easy-to-collect and readily available diagnostic fluid. Full article

Infectious bursal disease virus (IBDV) is one of the most important immunosuppressive viruses in poultry, causing the global spread of infectious bursal disease (IBD). It poses a significant threat to the healthy development of the poultry industry. Vaccination is an effective approach for controlling IBDV infection. Therefore, reliable immune monitoring for IBDV is critical for maintaining poultry health. The enzyme-linked immunosorbent assay (ELISA) is a common technique used to detect specific antibodies in clinical serum testing and for the serological evaluation of IBDV vaccines. Among the currently available and under development IBDV vaccines, IBD VP2 subunit-based vaccines account for a considerable proportion. These vaccines stimulate the production of antibodies that are specific only to VP2. However, most IBDV antibody ELISA kits approved for use have applied the whole virus as the coating antigen, which does not adequately meet the diverse requirements for IBDV detection across different conditions. This study utilized a prokaryotic expression system to express the VP2 protein of the IBDV epidemic strain, assembling it into virus-like particles to be used as coating antigens. This approach enabled the establishment of an indirect ELISA method for detecting IBDV VP2 antibody (VP2-ELISA). The optimal coated antigen concentration was determined to be 2.5 μg/mL, with overnight coating at 4 °C; sealing with 5% skim milk at 37 °C for 4 h; serum dilution at 1:500 with incubation at 37 °C for 30 min; secondary antibody dilution at 1:4000 with incubation at 37 °C for 40 min; and then incubation with the substrate solution 3,3′,5,5′-tetramethylbenzidine at room temperature for 20 min. The criterion for interpreting the detection results was OD_450nm ≥ 0.111 indicates IBDV antibody positivity, while OD_450nm < 0.111 indicates negativity. The established VP2-ELISA can specifically detect IBDV-positive sera at the lowest serum dilution of 1:6400, with intra- and inter-batch coefficients of variation of <2%. This indicates that the VP2-ELISA exhibits good specificity, sensitivity, and stability. Detection experiments using 20 laboratory-immunized chicken serum samples and 273 clinical serum samples demonstrated that the results of VP2-ELISA were consistent with those of commercial ELISA kits coated with whole virus. In summary, the VP2-ELISA developed in this study offers advantages in immune response detection for IBD VP2 subunit-based vaccines and is appropriate for evaluating the efficacy of IBD vaccines and detecting clinical serum samples. Full article

Background/Objectives: Porcine circovirus type 2d (PCV2d) is the predominant genotype associated with porcine circovirus-associated disease (PCVAD), leading to significant economic losses. In South Korea, current vaccine lot-release testing relies on a T/C-ratio-based guinea pig assay, which lacks scientific justification and methodological robustness. This study aimed to develop and validate a statistically defined in-house ELISA using rabbit-derived polyclonal antibodies against PCV2d for the standardized evaluation of immunogenicity. Methods: Polyclonal IgG was generated by immunizing a rabbit with inactivated PCV2d, and it was purified through Protein A chromatography. Guinea pigs (n = 18) were immunized with IMMUNIS^® DMVac, an inactivated PCV2d vaccine candidate developed by WOOGENE B&G, at different doses. In-house ELISA parameters were optimized (antigen coating, blocking agent, and substrate incubation), and analytical performance was evaluated by ROC, linearity, reproducibility, and specificity. Sera from guinea pigs and pigs were analyzed under validated conditions. Results: The optimal performance was achieved using 10⁵ genomic copies/mL of the antigen coating and a 5% BSA blocking agent. The assay showed strong diagnostic accuracy (AUC = 0.97), reproducibility (CVs < 5%), and linearity (R² = 0.9890). Specificity tests with PCV2a, PCV2b, and PRRSV showed minimal cross-reactivity (<7%). The cross-species comparison revealed a positive correlation (R² = 0.1815) and acceptable agreement (bias = −0.21) between guinea pig and porcine sera. The validated cut-off (S/P = 0.4) enabled accurate classification across both species and aligned well with commercial kits. Conclusions: The in-house ELISA offers a robust, reproducible, and scientifically validated platform for immunogenicity verification, supporting its application in Korea’s national lot-release system. Homologous competition assays with PCV2d are planned to further confirm antigen specificity. Full article

Steatotic liver disease is a global health challenge that requires reliable and noninvasive diagnostic biomarkers. This research aimed to validate the analytical and clinical performance of a fibroblast growth factor 21 (FGF21) enzyme-linked immunosorbent assay (ELISA) kit using an automated immunoassay analyzer. Plasma FGF21 levels were measured using a commercial ELISA kit on an automated immunoassay analyzer. Validation included intra- and inter-assay precision, dilution linearity, spike recovery, lower limit of quantification (LLOQ), interference testing, and sample stability analysis. Clinical evaluation involved 97 patients who underwent abdominal ultrasound-based attenuation imaging for the diagnosis of hepatic steatosis. The assay demonstrated high analytical precision, with intra- and inter-assay coefficients of variation <15% and an LLOQ of 3.260 pg/mL. Dilution linearity, spike recovery, and interference tests confirmed the reliability of the assay, whereas stability tests highlighted the minimal effect of freeze-thaw cycles and storage conditions. Clinically, FGF21 levels correlated with attenuation coefficient (r = 0.44). Diagnostic performance indicated 84% sensitivity and 81% specificity at defined FGF21 thresholds for the diagnosis of hepatic steatosis. This research confirmed the reliable analytical and clinical performance of the FGF21 ELISA kit, reinforcing its potential as a diagnostic biomarker of hepatic steatosis. Full article

Background: Several targeted drugs have been recently approved for the treatment of PIK3CA-mutated hormone receptor-positive (HR+)/HER2-negative (HER2−) breast cancer (BC). This study aimed at a comprehensive evaluation of the spectrum of PIK3CA alterations in Russian BC patients. Methods: The tumor material from 1872 patients with ER+/HER2− BC was tested by a combination of PCR-based methods. Results: Mutations were detected in 693/1872 (37%) cases, including 46 BC with two PIK3CA lesions. The three most common substitutions (E542K, E545K, and H1047R) were identified in 542/693 (78%) PIK3CA-mutated cases, while as many as 5.5–12% of identified mutations were not potentially detectable by common commercial kits. The study included patients of Slavic and non-Slavic ethnicities residing in regions with different climate conditions, however, these factors did not influence the distribution of PIK3CA mutations. The presence of PIK3CA variants was associated with older patient age at diagnosis (p = 0.0002), smaller tumor size (p = 0.005), lower grade (p = 0.005), Ki67 <20% (p = 0.0001) and progesterone receptor-positive status (p = 0.002) at the initial disease diagnosis, and fewer distant metastases at the time of the detection of BC spread (p = 0.0001). In a subgroup of 413 BC patients who received adjuvant tamoxifen or aromatase inhibitors, PIK3CA mutations were not associated with resistance to either type of treatment. Conclusions: The results of this study highlight the need to extend the PIK3CA testing beyond the hotspot regions of this gene. Although PIK3CA alterations contribute to the pathogenesis of HR+/HER2− BC and represent a target for several novel drugs, they are not intrinsically associated with unfavorable clinical characteristics of this subtype of cancer disease. Full article

Toxoplasma gondii is a globally significant zoonotic pathogen responsible for severe parasitic diseases in humans and animals. This study aimed to design, develop, and evaluate a novel immunochromatographic test (ICT) using a recombinant MIC2-MIC3 fusion protein (rMIC2-MIC3) for detecting specific antibodies against T. gondii. The ICT demonstrated exceptional sensitivity, capable of detecting T. gondii-specific antibodies in sera diluted up to 1:8. Specificity evaluation confirmed no cross-reactivity with antibodies against other parasites, such as Neospora caninum, Cryptosporidium suis, Eimeria tenella, and Sarcocystis tenella. Stability tests revealed the test strips maintained full functionality after 12 weeks of storage at 24 °C. The coincidence rate of the colloidal gold test strips prepared in this study with a commercial ELISA kit was 94.59%. Comparisons with advanced serodiagnostic tools, such as chimeric antigen-based ELISAs and recombinant protein diagnostics, further highlighted its robustness and applicability. These findings underscore the potential of the rMIC2-MIC3-based ICT as a reliable, economical, and accessible diagnostic tool for toxoplasmosis in veterinary and human medicine. Full article

Until recently, it was believed that bacterial translocation occurs as a result of leaky gut syndrome or sepsis. To confirm or exclude the process of bacterial translocation, biomarkers can be used. One such biomarker is defensins, which indicate immune activity, as defensins are cationic peptides with antibacterial properties produced by intestinal epithelial cells. Also, fatty acid-binding proteins (I-FABP and L-FABP) can serve as useful serological markers for intestinal epithelial damage, indicating impaired intestinal permeability or organ damage, as high concentrations of them are found in tissues and low concentrations in blood serum. In the context of obesity, the integrity of the intestinal barrier, which can be disrupted by dietary fat, leads to increased intestinal permeability. Since bacterial translocation and microbiota contribute to obesity and Metabolic Dysfunction-Associated Steatotic Liver Disease (MASLD) associated with metabolic dysfunction, intestinal barrier markers can be used to study the role of the gut–liver axis. The aim of this study was to gain insight into the pathogenesis of MASLD and examine the impact of bacterial translocation markers and intestinal and hepatic fatty acid-binding proteins (I-FABP and L-FABP) in children with MASLD. Method: We examined 60 children with MASLD and overweight/obesity (MASLD was diagnosed based on increased liver echogenicity in ultrasound and elevated ALT activity), aged 14.5 years (range 8.5 to 15.8); 33 children with overweight/obesity without MASLD, aged 13.0 years (range 11.4 to 15.8); and 16 healthy controls aged 11.0 years (range 7.0 to 16.2). Defensin, I-FABP, and L-FABP levels were measured using commercial kits: ELISA kits (Drg Medtek) were used to assess α-5 and α-6 defensin concentrations (HBD5, HBD6). I-FABP and L-FABP concentrations were measured using commercial ELISA kits (Hycult Biotech Inc., Wayne, PA, USA). ANOVA analysis was used to compare results across the three study groups. Results: A significant difference was found for the following tests among children with MASLD, obesity, and healthy controls: defensin 6 (14.4 ng/mL vs. 6.13 ng/mL vs. 17.2 ng/mL, respectively), L-FABP (9168 pg/mL vs. 7954 pg/mL vs. 7620 pg/mL, respectively), and I-FABP (272 pg/mL vs. 321 pg/mL vs. 330 pg/mL, respectively). No differences were found in defensin 5 levels (median 567.2 pg/mL vs. 485.7 pg/mL vs. 601.8 pg/mL). No differences were observed in cholesterol levels (HDL, LDL) or triglyceride concentrations, as well as apolipoprotein levels. Conclusions: Based on our study, it was concluded that inflammation and intestinal barrier damage lead to increased L-FABP levels, as it is released from enterocytes in response to oxidative stress or tissue damage. Defensin 6 may indirectly affect L-FABP through microbiota regulation and protection of the intestinal barrier. Defensin 6 also exerts antimicrobial activity and may accompany liver inflammation, with its increased concentration in comparison to obesity explained by the activation of defense mechanisms. Full article

Standard serological tests post-vaccination, such as enzyme-linked immunosorbent assay (ELISA), complement fixation, and virus neutralization, are crucial for monitoring African horse sickness (AHS). However, the availability of commercial test kits such as blocking ELISA varies by regions; while they are commonly used in Africa and Europe, their limited availability and high cost in Thailand present significant challenges. Therefore, this study aimed to evaluate an alternative approach using an in-house indirect ELISA based on cell-based monovalent and polyvalent strains of live attenuated AHS virus. This method addresses the cost and accessibility issues faced in Thailand. This study demonstrated promising results: the in-house indirect ELISA showed analytical sensitivity and specificity values of 88.30% and 67.02% for monovalent strains and 87.23% and 84.04% for polyvalent strains, respectively, compared to the blocking ELISA. These findings underscore the efficacy of the in-house ELISA as a viable serodiagnostic tool for AHS. Furthermore, the polyvalent antigen-based in-house indirect ELISA proved to be a reliable alternative for AHS monitoring, particularly in vaccinated horses, offering enhanced specificity. Additionally, this method is simpler, cheaper, faster, and more convenient than blocking ELISA and serum neutralization tests, making it a practical choice for routine AHS surveillance. Full article

Background: Tuberculosis (TB) remains a significant global health challenge, necessitating rapid and reliable diagnostic solutions. Current methods such as microscopy, culture, and PCR have limitations in resource-limited settings due to high costs and infrastructure requirements. Methods: In this study, we developed the MTB/IC LAMP-MS assay, a novel molecular diagnostic platform that integrates loop-mediated isothermal amplification (LAMP) technology with microscope (MS) detection. The assay enables TB diagnosis within 40 min, from nucleic acid extraction to result, requiring minimal equipment and simple operation for rapid and accurate detection. Results: Compared to the commercial AdvanSure TB/NTM real-time PCR kit, the MTB/IC LAMP-MS assay had a lower analytical sensitivity, with a limit of detection of 10³ CFU/mL versus 10² CFU/mL for the commercial PCR kit, but showed a sensitivity of 93.3% and specificity of 100% in clinical tests. Additionally, the assay demonstrated minimal cross-reactivity with other respiratory pathogens, underscoring its robust specificity. In addition, the preloaded microchips demonstrated stable performance for up to 12 weeks at room temperature, supporting their field applicability. Conclusions: With its lower cost, simplified operation, and minimal infrastructure requirements, the MTB/IC LAMP-MS assay provides a practical and efficient solution for TB diagnosis in resource-limited settings. Full article

Background: Respiratory Syncytial Virus (RSV) is a significant cause of morbidity and mortality among lung transplant (LTx) recipients. Therapeutic options are limited, emphasizing the importance of prevention. The Arexvy^® vaccine (RSVPreF3) showed promising efficacy among immunocompetent adults; however, data on its immunogenicity in solid organ transplant recipients remain unclear. Methods: A single-center retrospective cohort study, including all LTx recipients who were vaccinated with Arexvy in February 2024. Baseline and follow-up serum samples (1, 3, and 6 months post-vaccination) were analyzed for antibody responses using a commercial RSV ELISA kit and micro-neutralization assays against historical reference RSV A/B ATCC strains and seasonal RSV strains. Adverse events were documented. Results: A total of 28 recipients received the vaccine. Twenty-one (75%) were male, and the median age was 62 years (interquartile range [IQR], 53–67). The median time from transplant was 486 days (IQR, 243–966). Vaccination elicited strong immunogenic responses, demonstrating a twofold increase in ELISA-determined antibody levels at one month post-vaccination, which were sustained for six months. At one month, 67% of recipients had antibody levels exceeding the cutoff threshold. Micro-neutralization assays showed a significant increase in neutralizing antibodies against all tested variants (RSV A/B ATCC and seasonal RSV A/B), with titers remaining at least twofold higher than pre-vaccination levels. No serious adverse events were observed. Conclusions: Our findings demonstrate a sustained antibody response to the Arexvy^® vaccine in a cohort of LTx recipients, with antibody titers sustained over six months. Further research is needed to assess the long-term durability of the immune response and the potential immunogenicity of this vaccine in LTx populations. Full article

Background: The alarming increase in carbapenemase-producing Enterobacterales is a matter of grave public health concern. The most ubiquitous carbapenemases, Klebsiella pneumoniae carbapenemase (KPC)-, New Delhi metallo-β-lactamase (NDM)-, and oxacillinase (OXA-48)-like enzymes, belong to the Ambler molecular classes A, B, and D, respectively. KPC- and OXA-48-like enzymes have a serine-based hydrolytic mechanism, while NDMs are metallo-β-lactamases that contain zinc in the active site. For the judicious use of reserve drugs and promoting antimicrobial stewardship, timely detection of carbapenemases is essential. While molecular tools are the gold standard for the detection of these enzymes, many laboratories have limited access to them. This study focused on evaluating in-house tools and commercial phenotypic tests for the detection of OXA-48-, KPC-, and NDM-like enzymes in K. pneumoniae, the predominant extremely drug-resistant pathogen in Oman. Methods: In total, 80 GeneXpert/PCR-confirmed (40 OXA-48 and 20 KPC and NDM each) and 37 whole-genome-sequenced (25 OXA-232 and 6 KPC-2, plus NDM-1 and NDM-5) K. pneumoniae were subjected to screening by temocillin (30 μg disk) (MAST Diagnostica, Germany) and D71C (MASTDISCS^®). Isolates resistant to temocillin (<11 mm) and D71C were subjected to four tests: an in-house tool (OXA-48 disk test) and three commercial phenotypic tests: (i) the MASTDISCS^® Combi (D72C) (MAST Group Ltd., Bootle, UK); (ii) the MASTDISCS^® Combi (D73C) (MAST Group Ltd., UK); and (iii) an immunochromatographic assay (ICT), which is the KPC/IMP/NDM/VIM/OXA-48 Combo test kit (Medomics, China), for the detection of OXA-48-, KPC-, and NDM-like carbapenemases. Results: Temocillin exhibited good sensitivity and specificity (100% and 97.50%) compared to D71C (70% and 100%). Among the confirmatory tests, the in-house OXA-48 disk test had 92.50% sensitivity and 100% specificity, while the commercial MAST DISC tests D72C, D73C, and ICT had 97.50%, 95.00%, and 100% sensitivity and 100%, 91.67%, and 95% specificity, respectively. Conclusions: The temocillin disk test is a good screening tool. With high sensitivity and specificity, ease of performance, short turnaround time, and low cost, we recommend the ICT format for routine diagnostic use. In resource-constrained centers, the OXA-48 disk test is an excellent alternative with high sensitivity and specificity. Full article