Search Results (145)

Background/Objectives: The impact of antimicrobial susceptibility testing methodology on the categorization and positioning of cefiderocol and aztreonam-avibactam against metallo-β-lactamase (MBL)-producing Gram-negative bacilli remains unclear. This study aimed to evaluate the in vitro activity of cefiderocol and aztreonam-avibactam against clinical MBL-producing isolates and to assess the agreement between different cefiderocol susceptibility testing methods. Methods: A total of 299 non-duplicate clinical MBL-producing Gram-negative isolates were collected from clinical samples between 2022 and 2025. Antimicrobial susceptibility testing was performed using broth microdilution, disc diffusion, and gradient strip diffusion according to European Committee on Antimicrobial Susceptibility Testing (EUCAST) criteria. Carbapenemase genes were identified by immunochromatography and multiplex PCR. Categorical agreement and error rates between cefiderocol testing methods were analyzed. Results:Klebsiella pneumoniae was the predominant species, mainly producing NDM alone or in combination with OXA-48-like carbapenemases. Aztreonam-avibactam demonstrated complete activity against all Enterobacterales isolates (262/262, 100%) and high activity against Pseudomonas spp. (33/37, 89%). Cefiderocol susceptibility among Enterobacterales varied markedly depending on the testing method. Disc diffusion yielded 14% susceptibility (37/262), which increased to 52% (136/262) after ATU resolution, whereas broth microdilution showed 85% susceptibility (224/262). This resulted in low categorical agreement (42%) and a high rate of major errors (58%), with no very major errors detected. Cefiderocol activity did not differ substantially across carbapenemase types and was highest against VIM-producing Pseudomonas spp. Conclusions: Aztreonam-avibactam showed consistent in vitro activity against MBL-producing Enterobacterales, whereas cefiderocol activity was strongly influenced by the susceptibility testing methodology. Disc diffusion substantially underestimated cefiderocol susceptibility compared with broth microdilution. These findings highlight the critical impact of testing methodology on cefiderocol categorization and support the therapeutic role of last-line agents in the management of MBL-producing Gram-negative infections, with direct implications for clinical microbiology laboratories and antimicrobial stewardship programs. Full article

Background: Human cysticercosis, caused by the larval stage (cysticerci) of the pork tapeworm Taenia solium, is an important zoonotic disease. The disease is prevalent in developing countries where porcine cysticercosis is common and undercooked pork is habitually consumed. Objective: This study aimed to develop an immunochromatography-based test kit for the rapid diagnosis of human cysticercosis using low-molecular-weight antigens purified from cyst fluid of the T. solium Asian genotype to detect specific IgG antibodies in whole blood. The kit was designated as “the cysticercosis whole-blood test kit (iCys WB kit).” Methods: It was evaluated under laboratory conditions using 164 whole-blood samples, of which 21 were from confirmed cysticercosis cases. The results of the iCys WB kit, which detects anti-T. solium (cysticercus) antibodies in simulated whole blood samples, were compared with results from corresponding human serum samples. Results: When using both sample types, iCys WB kit demonstrated an accuracy of 98.8%, a sensitivity of 91.7%, a specificity of 100%, a positive likelihood ratio of 0, a negative likelihood ratio of 0.083, and an ROC area of 0.96. The agreement between results obtained from simulated whole-blood and serum samples showed perfect concordance. Conclusions: The iCys WB kit is a valuable easy-to-handle diagnostic tool and may be applicable for supporting clinical diagnosis at the point of care. Full article

Tetanus toxin evaluation has traditionally relied on mouse LD₅₀ bioassays, which require extensive animal use and time, necessitating development of alternative methods in accordance with 3R principles (Replacement, Reduction, and Refinement). We developed and validated a sandwich enzyme-linked immunosorbent assay (ELISA) as an alternative to animal testing for evaluating tetanus toxin biological activity using 18 environmental and clinical isolates of Clostridium tetani, complemented by an immunochromatographic (IC) assay for rapid screening. The ELISA demonstrated excellent analytical performance with a lower limit of quantification of 2.4 ng/mL (equivalent to 85.4 LD₅₀/mL), favorable linearity (R² = 0.999), precision (CV < 1.7–8.2%), and specificity (<1% cross-reactivity with C. septicum, C. novyi, and C. perfringens). Correlation analysis between ELISA relative potency and observed minimum lethal dose values revealed a robust positive correlation (r = 0.974). Both parallel line assay and single-point quantification methods showed strong correlations with mouse bioactivity measurements (r = 0.998). The IC assay successfully detected all isolates within 15 min. The measurement range of 2.4–45.6 ng/mL effectively covered diverse toxin-production capabilities spanning a 600-fold concentration range. This validated ELISA and IC assay combination provides a reliable, rapid alternative to animal experimentation for tetanus toxin evaluation. Full article

Potato virus X (PVX) and potato virus Y (PVY) are major pathogens that threaten seed potato quality and yield. To improve the efficiency of field screening, we developed monovalent PVX, monovalent PVY, and bivalent PVX/PVY nanozyme strips using Fe₃O₄ nanozymes as labels in a double-antibody sandwich lateral flow immunochromatographic assay. Western blot analysis demonstrated that four monoclonal antibodies (PVX 2, PVX 6, PVY 2, and PVY 5) specifically recognized their corresponding viral coat proteins. Specificity testing showed that the nanozyme strips reacted only with the target viruses and did not cross-react with other common potato viruses, including Potato virus A (PVA), Potato virus M (PVM), Potato virus S (PVS), and Potato leafroll virus (PLRV). The PVX nanozyme strip detected PVX-positive extracts diluted up to 10³-fold, the PVY nanozyme strip up to 10⁴-fold, and the bivalent strip detected PVX/PVY co-infected samples diluted up to 10³-fold. In addition, detection results by strips from 12 samples of plantlets in vitro were fully consistent with RT-PCR. These nanozyme strips provide rapid, simple, specific, and sensitive methods that can be stored at ambient temperature, enabling field surveys, warehouse screening, and on-site testing and supporting early detection of potato virus diseases. Full article

Background: Scrub typhus is commonly misdiagnosed because of nonspecific clinical features and limited data on the performance of diagnostic tests. This study aimed to evaluate the accuracy of commercially available serological and molecular assays for diagnosing scrub typhus. Methods: Adult patients with suspected scrub typhus who visited a tertiary-care hospital in South Korea from July 2022 to December 2024 were prospectively enrolled. Scrub typhus was confirmed by either a positive real-time polymerase chain reaction (PCR) result for Orientia tsutsugamushi or a ≥ four-fold increase in the O. tsutsugamushi-specific total immunoglobulin (Ig) antibody titer on an immunofluorescence assay (IFA). The diagnostic performances of the serial IFA, an immunochromatography-based rapid diagnostic test (ICT), and multiplex real-time PCR targeting the groEL and 47-kDa genes were compared. Results: Among 159 patients, 81 had scrub typhus and 78 did not. The sensitivity and specificity were 64% and 100% for the serial IFA, 75% and 91% for the ICT, and 95% and 100% for multiplex PCR, respectively. The area under the curve was significantly higher for the ICT (0.819) than for the acute-phase IFA (0.743, p = 0.02). Conclusions: Multiplex real-time PCR provided rapid and highly accurate confirmation of scrub typhus, and an acute-phase ICT may be an alternative to a single acute-phase IFA for early clinical decision-making. Full article

Background/Objectives: Viral gastroenteritis leads to a broad range of hospitalization costs globally in children, depending on the region. To our knowledge, no recent studies have examined the hospitalization cost associated with viral gastroenteritis in Romania. The aim of this study is to determine the direct hospitalization cost of community-acquired viral gastroenteritis (rotavirus, adenovirus and norovirus) in children admitted to Children’s Clinical Hospital of Brașov, Romania, for one year. Methods: All children aged 0–60 months hospitalized for a stool sample positive for rotavirus, adenovirus or norovirus during January 2023 and December 2023 were included in this study. Hospital-acquired gastroenteritis, gastrointestinal coinfections or children with acute coinfection were excluded. The stool specimens were tested using the immunochromatography method. Results: Out of the total of 282 children, 218 children presented rotavirus gastroenteritis, 35 children presented adenovirus gastroenteritis and 29 children presented norovirus gastroenteritis. Regarding patient characteristics, a higher proportion of boys than girls was observed in all three comparison groups, the average age for children with rotavirus was 22.2 months vs. norovirus and adenovirus, and children presented an average age of 16.4 months. Average hospitalization length of stay for rotavirus was 4.64 (±1.95) days, for adenovirus it was 4.54 (±1.52) days and for norovirus it was 4.75 (±1.93) days. Direct hospitalization costs did not differ between rotavirus, adenovirus, and norovirus infections (Kruskal–Wallis H(2) = 0.145, p = 0.930). Conclusions: In this single-center study, rotavirus remained the most frequent cause of viral gastroenteritis requiring hospitalization in young children, followed by adenovirus and norovirus. Although the average length of stay was similar across groups, hospitalization costs varied, with rotavirus-associated cases showing the highest mean expenses and widest cost variability. Full article

It is crucial to identify Enterobacterales that produce carbapenemase to treat and manage hospital infections. The suggested techniques for their identification need a lengthy wait, technical knowledge, and training. Lateral flow immunoassays (LFIAs) provide a solution to these requirements. Thus, this study compared LFIA with phenotypic and genotypic tests for carbapenemase-producing bacteria. Fifty clinical isolates of carbapenem-resistant superbugs were examined. KPC, VIM, NDM, IMP, and OXA-48-like enzymes were evaluated and compared with phenotypic tests and LFIA. Regarding the phenotypic characteristics, the mCIM was positive in 37/50 (74%), and the eCIM was positive in 21/50 (42%). Regarding using LFIA, 41 out of the total isolates (82%) gave a positive red line with one or more of the tested genes. The most frequently detected gene was bla_NDM (27/50 (54%)), and the least detected one was bla_IMP (14/50 (28%)), which was in accordance with the PCR results. While investigating the accuracy of LFIA vs. PCR, it was found that LFIA had 100% sensitivity in the detection of the bla_NDM and bla_OXA genes, with 85.2% and 91.4% specificity, respectively, while for the bla_IMP, bla_KPC, and bla_VIM genes, the values were 91.7% and 92.1%, 94.1% and 90.9%, and 95.5% and 89.3%, respectively. The overall accuracy of LFIA ranged from 92 to 94%. Our comparison with molecular assays revealed remarkable agreement, so we propose that this test might be utilized as a supplementary tool. Full article

The four serotypes of dengue virus (DENV), types 1 to 4 (DENV-1 to DENV-4), exhibit approximately 60% identity in the encoded amino acid residues of viral proteins. Reverse transcription of RNA extracted from patient serum specimens followed by PCR amplification with serotype-specific probes is the current standard technique for DENV serotyping. However, this method is time- and cost-consuming, and rapid detection systems with low cost are desirable. Previously, we developed a prototype serotype-specific immunochromatography system. That system was composed of four strips with four corresponding distinct sample buffers, each specifically detecting a single DENV serotype. In the present study, we improved this system by combining pairs of strips into one lateral-flow cassette each, providing DENV-1 and DENV-2 detection in one device and DENV-3 and DENV-4 detection in a second device; this strategy successfully reduced the required sample volume. Furthermore, we were able to adjust the composition of the sample buffers such that a single sample buffer sufficed for all four DENV serotype detection reactions, allowing much easier handling of the devices. Evaluation of this new device against laboratory and clinical DENV isolates and clinical specimens from DENV-infected individuals showed sensitivity that was comparable to that of our previous version, yielding serotype specificity of 100%. These new devices are expected to be of use in the clinical setting, accelerating both prospective and retrospective epidemiological studies. Full article

Carbapenem-resistant Klebsiella pneumoniae isolates harbouring double carbapenemases, from patients in a surgical and transplantation ICU, were investigated to better understand the dispersion of the pathogen. Twenty-three carbapenem-resistant K. pneumoniae isolates harbouring at least two different carbapenemases (by immunochromatography screening), were consecutively collected during a seven-month period from patients in a surgical and transplantation ICU. Identification and susceptibility testing were performed using the MALDI-TOF Vitek MS and the Vitek2 system (BioMerieux), respectively. Whole genome sequencing (WGS) was performed in an Illumina NextSeq2000 platform and MLST and resistome analysis of assembled genomes were performed by ResFinder, through the Center for Genomic Epidemiology platform. All isolates were resistant to ertapenem, imipenem, meropenem, and most to meropenem–varbobactam. Seventeen isolates belonged to the ST11 type and were positive for the OXA-48/NDM combination (by immunochromatography and NGS). Four isolates belonged to the ST39 type and were positive for the KPC/NDM combination (by immunochromatography and NGS). Finally, two isolates belonged to the ST258 type. One of them was positive for the OXA-48/KPC/NDM combination (by immunochromatography), but only bla_KPC was detected by WGS, and the second was positive for the OXA-48/KPC combination (by immunochromatography) and confirmed by WGS. This is the first report of an outbreak in Greece due to two simultaneous carbapenem-resistant populations harbouring double carbapenemases: a larger one comprising ST11 isolates harbouring the combination bla_NDM-1/bla_OXA-48, coupled by a smaller one comprising ST39 isolates harbouring the combination bla_KPC-2/bla_NDM-1. The implications of this particular situation regarding public health as well as intra-nosocomial infection prevention and control should be further monitored and studied. Full article

Beta-agonists are growth promoters sparking considerable interest in animal husbandry. However, their numerous negative effects on health have led to a number of restrictions on their presence in agricultural products, which differ depending on the type of preparation and food. In this regard, there is a demand for methods of their mass, rapid, and easy control with strict, focused selectivity. In this paper, a lateral flow immunoassay (LFIA) with clenbuterol (CLE), a priority β2-agonist for food safety, as the main target compound is proposed. The LFIA is based on indirect labeling of specific antibodies by gold nanoparticles via anti-species antibodies. The development of the LFIA involved optimizing the concentrations of immunoreagents and the composition of the reaction mixture (buffer type and pH, ionic strength, detergents, and additional components). In qualitative (visual) mode, the LFIA detects up to 1.0 ng/mL of CLE. In quantitative mode, the detection limit reaches 0.02 ng/mL, surpassing previously described colorimetric LFIAs. The selectivity of the obtained and used monoclonal antibodies allows for the group-specific detection of CLE and structurally close (the presence of a trimethyl residue, similar charge distribution in the benzene ring) common β2-agonists—salbutamol and mabuterol—distinguishing them from other β-agonists, including the widely used β1-agonist ractopamine, which differs in application and biological activity. The assay time is 15 min. The application of the LFIA for meat samples demonstrated that the CLE recovery ranged between 86% and 104%. The obtained results confirm the effectiveness and competitive potential of the developed assay for screening meat products outside of laboratories. Full article

Intestinal protozoa are a common cause of morbidity in people living with HIV (PWH), particularly in tropical regions with poor sanitation. We conducted a cross-sectional study in 315 PWH from Iquitos, Peru, between October 2023 and May 2024, to assess their prevalence and risk factors. Stool samples were examined using Lugol’s iodine, modified Ziehl–Neelsen (MZN) staining, and immunochromatography (ICT). The mean age was 41 years, with a median CD4+ count of 431 cells/µL; 12.4% were in the AIDS stage, and 21.5% had a detectable viral load. 51.4% of the participants tested positive for any intestinal protozoa. The overall Cryptosporidium spp. prevalence (by combining MZN and ICT results) was 25.7%. The overall Giardia spp. and Entamoeba spp. prevalences (by combining Lugol’s iodine and ICT results) were 2.9% and 1.9%, respectively. Blastocystis spp. was frequently isolated, though its pathogenicity remains uncertain. Diagnostic agreement was almost perfect between Lugol and ICT for Giardia and Entamoeba (κ = 0.87; p < 0.001 and κ = 0.91; p < 0.001, respectively), but only slight between MZN and ICT. Homosexual practices were identified as a significant risk factor for pathogenic protozoa infection (AOR 2.52; 95% CI: 1.04–6.12). In conclusion, the high prevalence of protozoa infection reflects ongoing fecal–oral exposure, underscoring the need for public health education, routine diagnosis, and treatment in similar settings. Full article

Respiratory syncytial virus (RSV) is a major cause of severe respiratory infections, particularly in infants and young children. Although disinfection methods using alcohol and detergents are effective, their application in pediatric environments poses safety concerns. Ozone (O₃) has been employed for water treatment, food preservation, and air purification, but its efficacy against RSV has not been well studied. Here, we investigated the inactivation of RSV using a dielectric barrier discharge (DBD)-based ozone generator (SFG1210). The RSV A2 strain was spotted on glass coverslips and exposed to low-concentration ozone (0.5 ppm) for 1 h under controlled temperature (24.6~27.2 °C) and relative humidity (71.9~75.1%) conditions. Subsequent infectivity assays combined with immunochromatography showed that ozone exposure significantly reduced RSV infectivity. Specifically, viral titration assay of median tissue culture infectious dose (TCID₅₀) showed that RSV titers were reduced by more than 6 logs. In addition, biochemical analyses showed significant reductions in intact RSV genomic RNA and F protein levels after ozone treatment, suggesting that ozone inactivates RSV by damaging both the viral genome and surface proteins. These findings demonstrate the potential applicability of the SFG1210 ozone generator as an effective tool for surface disinfection of RSV, providing a safe, non-contact, and practical approach for infection control in healthcare and childcare settings. Full article

In recent decades, the problem of resistant strains, which present resistance to different types of antimicrobials, has increased. Klebsiella pneumoniae is one of the most important species that exhibits an acquired resistance phenotype to at least one agent in three or more classes of antimicrobials and is thus characterized as a multidrug-resistant bacterium (MDR). 98 nosocomial strains of K. pneumoniae were isolated during the pre-COVID-19 period, and more specifically, from February 2015 to March 2019, were analyzed for the detection of class A, D, and B carbapenemase genes. The existence of KPC, OXA-48 like, IMP, VIM, and NDM carbapenemases has been examined. The immunochromatography showed that NDM carbapenemases are more frequently detected in the samples, reaching a percentage of 30.7%, while correspondingly the percentage for VIM carbapenemases was 7.68% among the strains with resistant phenotypes. No strain with carbapenemase IMP was found. Real-time multiplex polymerase chain reaction (PCR) showed, in contrast to immunochromatography kits, that a high percentage of bacterial isolates (94.26%) carry NDM and VIM carbapenemase genes, while no IMP carbapenemase genes were detected. Regarding the KPC enzymes, the immunochromatography kits showed that KPC positive strains are reaching 53.1%, and OXA-48 positive strains are reaching 3.1% among the strains with resistant phenotypes. Real-time multiplex polymerase chain reaction revealed a much higher percentage of 89.6% KPC positive isolates and a percentage of 14.6% OXA-48 carbapenemase producers. The aforementioned results indicate the dominance of the Multiplex Real-Time PCR as a “gold standard” method. This study could not fully support the usefulness of rapid immunochromatographic tests as a fast and useful diagnostic tool in the laboratory daily routine, as per the results of previous studies. Thus, more studies need to be conducted in this field to introduce these rapid tests safely into the daily laboratory workflow as a screening tool. Additionally, this study underlines the predominance of KPC enzymes from clinical isolates of ICUs and a significant shift over the OXA-48 like enzymes that are not limited to the ICU environment. Full article

Periodontitis is a biofilm-driven inflammatory disease in which conventional indices (probing depth, clinical attachment level, and radiographs) quantify tissue destruction without capturing the biology of infection. In this review, we synthesized microbiological diagnostics, from chairside tools to omics. We outline sampling strategies and emphasize the quantitative monitoring of bacterial load. Enzymatic assays (e.g., N-benzoyl-DL-arginine-2-naphthylamide hydrolysis assay test) measure functional activity at the point of care. Immunological methods include rapid immunochromatography for Porphyromonas gingivalis and enzyme-linked immunosorbent assay for the high-throughput measurement of bacterial antigens. Molecular platforms encompass quantitative polymerase chain reaction (qPCR) (TaqMan, SYBR, multiplex panels; propidium monoazide quantitative-qPCR for viable cells), checkerboard DNA–DNA hybridization for semi-quantitative community profiling, loop-mediated isothermal amplification (LAMP)/molecular beacon-LAMP for portable isothermal detection, and microarrays. Complementary modalities such as fluorescent in situ hybridization, next-generation sequencing, and Fourier transform infrared spectroscopy provide spatial, ecological, and biochemical resolutions. We discuss the limitations of current approaches, including sampling bias, presence–activity discordance, semi-quantitation, method biases, limited strain/function resolution, low-biomass artifacts, and lack of validated cutoffs. To address these challenges, we propose a pragmatic hybrid strategy: site-specific quantitative panels combined with activity and host-response markers interpreted alongside clinical metrics under standardized quality assurance/quality control. Priorities include outcome-linked thresholds, strain-aware/functional panels, robust point-of-care chemistry, and harmonized protocols to enable personalized periodontal care. Full article

Bovine viral diarrhea virus (BVDV) is an important pathogen in cattle and causes considerable economic losses worldwide. In Argentina, where there is no national control program, BVDV remains endemic. In this retrospective study, the epidemiological, clinical and pathological features of postnatal BVDV-associated diseases in 50 outbreaks in central Argentina (1995–2024) were analyzed. Data were obtained from field reports, necropsies, and virological results (virus isolation, RT-nPCR, immunochromatography). No seasonal pattern was found. Acute infections (AIs) and mucosal disease (MD) occurred with similar frequency. Clinical signs included salivation, weakness, emaciation and diarrhea. The lesions were widespread and involved the gastrointestinal tract, skin, lymphoid tissues and spleen. Although MD cases has more extensive tissue involvement, no significant differences in morbidity, mortality or distribution of lesions were observed between AIs and MD. BVDV-1b was the most frequently detected subtype. These results highlight the challenges of BVDV control in extensive production systems. Strengthening diagnostic surveillance, implementing targeted vaccination and eliminating persistently infected animals are essential to reduce BVDV impact in endemic regions such as Argentina. Full article