Search Results (18)

Background/Objectives: Phenotypic antimicrobial susceptibility testing (AST) is essential for guiding timely and effective antibiotic therapy. Rapid and accurate reporting of AST results enables earlier optimization of treatment and supports antimicrobial stewardship by minimizing unnecessary use of broad-spectrum antibiotics. This study aimed to evaluate the performance of the Selux DX Next-Generation Phenotyping AST system in comparison with the standard-of-care MicroScan WalkAway Plus system and broth microdilution reference results. Methods: A total of 332 clinical isolates and 97 Antimicrobial Resistance (AR) Bank reference isolates were tested using the Selux DX and MicroScan systems. Performance was assessed by categorical agreement (CA), error rates [very major errors (VMEs), major errors (MEs), minor errors (mEs)], and turnaround time. Results: The Selux DX system demonstrated ≥90% CA for most drug–organism combinations, consistent with Clinical and Laboratory Standards Institute (CLSI) acceptance thresholds, although elevated error rates were noted for erythromycin, aztreonam, cefazolin, minocycline, and ampicillin/sulbactam. Across 5124 drug–bug combinations, 55 VMEs (1.1%), 42 MEs (0.8%), and 203 mEs (4.0%) were identified. The Selux DX system achieved a markedly shorter average turnaround time of 5.5 h compared with 16 h for the MicroScan system, though at the cost of a longer setup time. Conclusions: The Selux DX system provides rapid and reliable phenotypic AST results, supporting earlier clinical decision-making and antimicrobial stewardship. However, discrepancies with certain antimicrobial agents, particularly among highly resistant reference isolates, highlight the need for further validation in larger, multicenter studies. Full article

Background: The issue of Helicobacter pylori (H. pylori) resistance to clarithromycin (CLR) has consistently posed challenges for clinical treatment. Hence, a rapid susceptibility testing (AST) method urgently needs to be developed. Methods: In the present study, 35 isolates of H. pylori were isolated from 203 gastritis patients of the Guangzhou cohort, and the antimicrobial resistance phenotypes were associated with their genomes to analyze the relevant mutations. Based on these mutations, a rapid detection system utilizing high-resolution melting (HRM) curve analysis was designed and verified by the Shenzhen cohort, which consisted of 38 H. pylori strains. Results: Genomic analysis identified the mutation of the 2143 allele from A to G (A2143G) of 23S rRNA as the most relevant mutation with CLR resistance (p < 0.01). In the HRM system, the wild-type H. pylori showed a melting temperature (Tm) of 79.28 ± 0.01 °C, while the mutant type exhibited a Tm of 79.96 ± 0.01 °C. These differences enabled a rapid distinction between two types of H. pylori (p < 0.01). Verification examinations showed that this system could detect target DNA as low as 0.005 ng/μL in samples without being affected by other gastric microorganisms. The method also showed a good performance in the Shenzhen validation cohort, with 81.58% accuracy, and 100% specificity. Conclusions: We have developed an HRM system that can accurately and quickly detect CLR resistance in H. pylori. This method can be directly used for the detection of gastric microbiota samples and provides a new benchmark for the simple detection of H. pylori resistance. Full article

Multidrug-resistant Candida auris has recently caused major outbreaks in healthcare facilities. Rapid and accurate antifungal susceptibility testing (AST) of C. auris is crucial for proper management of invasive infections. The Commercial Sensititre Yeast One and Vitek 2 methods underestimate or overestimate the resistance of C. auris to fluconazole and amphotericin B (AMB). This study evaluated the AST results of C. auris against fluconazole and AMB by gradient-MIC-strip (Etest) and broth microdilution-based MICRONAUT-AM-EUCAST (MCN-AM) assays. Clinical C. auris isolates (n = 121) identified by phenotypic and molecular methods were tested. Essential agreement (EA, ±1 two-fold dilution) between the two methods and categorical agreement (CA) based on the Centers for Disease Control and Prevention’s (CDC’s) tentative resistance breakpoints were determined. Fluconazole resistance-associated mutations were detected by PCR-sequencing of ERG11. All isolates identified as C. auris belonged to South Asian clade I and contained the ERG11 Y132F or K143R mutation. The Etest–MCN-AM EA was poor (33%) for fluconazole and moderate (76%) for AMB. The CA for fluconazole was higher (94.2%, 7 discrepancies) than for AMB (91.7%, 10 discrepancies). Discrepancies were reduced when an MCN-AM upper-limit value of 4 µg/mL for fluconazole-susceptible C. auris and an Etest upper-limit value of 8 µg/mL for the wild type for AMB were used. Our data show that resistance to fluconazole was underestimated by MCN-AM, while resistance to AMB was overestimated by Etest when using the CDC’s tentative resistance breakpoints of ≥32 µg/mL for fluconazole and ≥2 µg/mL for AMB. Method-specific resistance breakpoints should be devised for accurate AST of clinical C. auris isolates for proper patient management. Full article

The rapid rise in increasingly resistant bacteria has become a major threat to public health. Antimicrobial susceptibility testing (AST) is crucial in guiding appropriate therapeutic decisions and infection prevention practices for patient care. However, conventional culture-based AST methods are time-consuming and labor-intensive. Therefore, rapid AST approaches exist to address the delayed gap in time to actionable results. There are two main types of rapid AST technologies— phenotypic and genotypic approaches. In this review, we provide a summary of all commercially available rapid AST platforms for use in clinical microbiology laboratories. We describe the technologies utilized, performance characteristics, acceptable specimen types, types of resistance detected, turnaround times, limitations, and clinical outcomes driven by these rapid tests. We also discuss crucial factors to consider for the implementation of rapid AST technologies in a clinical laboratory and what the future of rapid AST holds. Full article

The MIC value definition faithfully reflects antimicrobial sensitivity, profoundly impacting the infection's clinical outcome. Our study aimed to evaluate the Accelerate Pheno^TM System in defining the importance of fast phenotypic susceptibility data. A number of 270 monomicrobial samples simultaneously underwent standard procedures and fast protocols after a contemporary Gram stain. Finally, we provided Turn-around Time (TAT) and statistical evaluations. The fast technology required a medium value of 7 h to complete ID and AST profiles. Although there were some spectrum limitations, it revealed an optimal success rate in microbial identification directly from positive blood cultures. The Gram-negative AST reached a 98.9% agreement between the Accelerate Pheno™ System and the standard method. In addition, the Gram-positive AST gathered a 98.7% agreement comparing the same systems. The chance to rapidly provide precise MIC values is one of the last frontiers in clinical microbiology, especially in high-prevalence antimicrobial resistance areas. Full article

Antimicrobial resistance (AMR) is a global threat fueled by incorrect (and overuse) of antibiotic drugs, giving rise to the evolution of multi- and extreme drug-resistant bacterial strains. The longer time to antibiotic administration (TTA) associated with the gold standard bacterial culture method has been responsible for the empirical usage of antibiotics and is a key factor in the rise of AMR. While polymerase chain reaction (PCR) and other nucleic acid amplification methods are rapidly replacing traditional culture methods, their scope has been restricted mainly to detect genotypic determinants of resistance and provide little to no information on phenotypic susceptibility to antibiotics. The work presented here aims to provide phenotypic antimicrobial susceptibility testing (AST) information by pairing short growth periods (~3–4 h) with downstream PCR assays to ultimately predict minimum inhibitory concentration (MIC) values of antibiotic treatment. To further simplify the dual workflows of the AST and PCR assays, these reactions are carried out in a single-vessel format (PCR tube) using novel lyophilized reagent beads (LRBs), which store dried PCR reagents along with primers and enzymes, and antibiotic drugs separately. The two reactions are separated in space and time using a melting paraffin wax seal, thus eliminating the need to transfer reagents across different consumables and minimizing user interactions. Finally, these two-step single-vessel reactions are multiplexed by using a microfluidic manifold that allows simultaneous testing of an unknown bacterial sample against different antibiotics at varying concentrations. The LRBs used in the microfluidic system showed no interference with the bacterial growth and PCR assays and provided an innovative platform for rapid point-of-care diagnostics (POC-Dx). Full article

Urinary tract infections (UTIs) are one of the most common human infections and are most often caused by Gram-negative bacteria such as Escherichia coli. In view of the increasing number of antibiotic-resistant isolates, rapidly initiating effective antibiotic therapy is essential. Therefore, a faster antibiotic susceptibility test (AST) is desirable. The MALDI-TOF MS-based phenotypic antibiotic susceptibility test (MALDI AST) has been used in blood culture diagnostics to rapidly detect antibiotic susceptibility. This study demonstrates for the first time that MALDI AST can be used to rapidly determine antibiotic susceptibility in UTIs directly from patients’ urine samples. MALDI-TOF MS enables the rapid identification and AST of Gram-negative UTIs within 4.5 h of receiving urine samples. Six urinary tract infection antibiotics, including ciprofloxacin, cotrimoxazole, fosfomycin, meropenem, cefuroxime, and nitrofurantoin, were analyzed and compared with conventional culture-based AST methods. A total of 105 urine samples from UTI patients contained bacterial isolates for MALDI AST. The combination of ID and AST by MALDI-TOF allowed us to interpret the result according to EUCAST guidelines. An overall agreement of 94.7% was found between MALDI AST and conventional AST for the urinary tract pathogens tested. Full article

The development of efficient point-of-care (POC) diagnostic tools for detecting infectious diseases caused by destructive pathogens plays an important role in clinical and environmental monitoring. Nevertheless, evolving complex and inconsistent antibiotic-resistant species mire their drug efficacy. In this regard, substantial effort has been expended to develop electrochemical sensors, which have gained significant interest for advancing POC testing with rapid and accurate detection of resistant bacteria at a low cost compared to conventional phenotype methods. This review concentrates on the recent developments in electrochemical sensing techniques that have been applied to assess the diverse latent antibiotic resistances of pathogenic bacteria. It deliberates the prominence of biorecognition probes and tailor-made nanomaterials used in electrochemical antibiotic susceptibility testing (AST). In addition, the bimodal functional efficacy of nanomaterials that can serve as potential transducer electrodes and the antimicrobial agent was investigated to meet the current requirements in designing sensor module development. In the final section, we discuss the challenges with contemporary AST sensor techniques and extend the key ideas to meet the demands of the next POC electrochemical sensors and antibiotic design modules in the healthcare sector. Full article

Bloodstream infections are a leading cause of morbidity and mortality. The rapid diagnostic testing of positive blood cultures (PBCs) shortens times to effective therapy and the de-escalation of broad-spectrum empiric therapy. This is the first study examining the Qvella FAST^TM System for the rapid (~20 min) purification of microorganisms directly from PBCs using BacT/Alert^® FA/FAN bottles in the bioMérieux Virtuo instrument. We compared the performance of the FAST^TM System Liquid Colony^TM (LC), for immediate downstream ID and phenotypic AST, to standard workflow involving colonies obtained by overnight subculture. The LC yielded a concordant species ID by VITEK MS in 121/138 (87.7%) samples, identifying 32 different Gram-positive and Gram-negative species with 3/123 (2.6%) discordances. Compared to standard workflow, direct AST of the LC using VITEK^® 2 yielded 98.4% categorical agreement and 98.0% essential agreement. Very major error, major error, and minor error rates were 1.0%, 0.0%, and 1.8%, respectively, for Gram-negative organisms; and 1.9%, 0.2%, and 1.2%, respectively, for Gram-positive organisms. The median times from positive blood culture flag to results by FAST^TM System for ID and AST were 7.8 h and 15.7 h, respectively, versus 22.4 h and 36.6 h for standard workflow, respectively. In conclusion, the FAST^TM System provides reliable results for direct ID and AST from PBCs with significantly decreased turnaround times. Full article

Accelerating antimicrobial susceptibility testing (AST) is a priority in the development of novel microbiological methods. The MALDI-TOF MS-based direct-on-target microdroplet growth assay (DOT-MGA) has recently been described as a rapid phenotypic AST method. In this proof-of-principle study, we expanded this method to simultaneously test 24 antimicrobials. An Enterobacterales panel was designed and evaluated using 24 clinical isolates. Either one or two (only for antimicrobials with the EUCAST “I” category) breakpoint concentrations were tested. Microdroplets containing bacterial suspensions with antimicrobials and growth controls were incubated directly on the spots of a disposable MALDI target inside a humidity chamber for 6, 8 or 18 h. Broth microdilution was used as the standard method. After 6 and 8 h of incubation, the testing was valid (i.e., growth control was successfully detected) for all isolates and the overall categorical agreement was 92.0% and 92.7%, respectively. Although the overall assay performance applying short incubation times is promising, the lower performance with some antimicrobials and when using the standard incubation time of 18 h indicates the need for thorough standardization of assay conditions. While using “homebrew” utensils and provisional evaluation algorithms here, technical solutions such as dedicated incubation chambers, tools for broth removal and improved software analyses are needed. Full article

Current methods for the diagnosis of urinary tract infections with antimicrobial susceptibility testing take 2–3 days and require a clinical laboratory. The lack of a rapid, point-of-care antibiotic susceptibility test (AST) has contributed to the misuse of antibiotics when treating urinary tract infections (UTIs) and consequently to the rise of multi-drug-resistant organisms. The current clinical approach has led to reduced treatment options and increased costs of diagnosis and therapy. To address this issue, novel diagnostics are needed for the timely determination of antimicrobial susceptibility. We present a rapid, point-of-care, phenotypic AST device that can report the antibiotic susceptibility/resistance of a uropathogen to a panel of antibiotics in as few as 3 h by utilizing fluorescent-labelling chemistry and a highly sensitive particle-counting instrument. We analysed 744 urine samples from the outpatients and inpatients of two Italian hospitals. The 130 UTI-positive patient urine samples we found were measured using a panel of six common UTI antibiotics plus a growth control. By comparing our results to hospital laboratory urine cultures, we obtained an overall sensitivity = 81%, a specificity = 83%, an SPV (sensitivity predicted value) = 95%, and an RPV (resistance predicted value) = 54%. According to our preliminary data, the sensitivity predicted value for a single antibiotic agent was 95%, thus allowing (in the vast majority of cases) an early (within 3 h) recognition of an effective agent for a single patient. Full article

The indiscriminate use and mismanagement of antibiotics over the last eight decades have led to one of the main challenges humanity will have to face in the next twenty years in terms of public health and economy, i.e., antimicrobial resistance. One of the key approaches to tackling antimicrobial resistance is clinical, livestock, and environmental surveillance applying methods capable of effectively identifying antimicrobial non-susceptibility as well as genes that promote resistance. Current clinical laboratory practices involve conventional culture-based antibiotic susceptibility testing (AST) methods, taking over 24 h to find out which medication should be prescribed to treat the infection. Although there are techniques that provide rapid resistance detection, it is necessary to have new tools that are easy to operate, are robust, sensitive, specific, and inexpensive. Chemical sensors and biosensors are devices that could have the necessary characteristics for the rapid diagnosis of resistant microorganisms and could provide crucial information on the choice of antibiotic (or other antimicrobial medicines) to be administered. This review provides an overview on novel biosensing strategies for the phenotypic and genotypic determination of antimicrobial resistance and a perspective on the use of these tools in modern health-care and environmental surveillance. Full article

Pneumonic plague is a lethal infectious disease caused by Yersinia pestis, a Tier-1 biothreat agent. Antibiotic treatment can save infected patients; however, therapy should begin within 24 h of symptom onset. As some Y. pestis strains showed an antibiotic resistance phenotype, an antibiotic susceptibility test (AST) must be performed. Performing the Clinical and Laboratory Standards Institute (CLSI)-recommended standard process, which includes bacterial isolation, enumeration and microdilution testing, lasts several days. Thus, rapid AST must be developed. As previously published, the Y. pestis-specific reporter phage ϕA1122::luxAB can serve for rapid identification and AST (ID-AST). Herein, we demonstrate the ability to use ϕA1122::luxAB to determine minimal inhibitory concentration (MIC) values and antibiotic susceptibility categories for various Y. pestis therapeutic antibiotics. We confirmed the assay by testing several nonvirulent Y. pestis isolates with reduced susceptibility to doxycycline or ciprofloxacin. Moreover, the assay can be performed directly on positive human blood cultures. Furthermore, as Y. pestis may naturally or deliberately be spread in the environment, we demonstrate the compatibility of this direct method for this scenario. This direct phage-based ID-AST shortens the time needed for standard AST to less than a day, enabling rapid and correct treatment, which may also prevent the spread of the disease. Full article

Due to the inappropriate use and overuse of antibiotics, the emergence and spread of antibiotic-resistant bacteria are increasing and have become a major threat to human health. A key factor in the treatment of bacterial infections and slowing down the emergence of antibiotic resistance is to perform antimicrobial susceptibility testing (AST) of infecting bacteria rapidly to prescribe appropriate drugs and reduce the use of broad-spectrum antibiotics. Current phenotypic AST methods based on the detection of bacterial growth are generally reliable but are too slow. There is an urgent need for new methods that can perform AST rapidly. Bacterial metabolism is a fast process, as bacterial cells double about every 20 to 30 min for fast-growing species. Moreover, bacterial metabolism has shown to be related to drug resistance, so a comparison of differences in microbial metabolic processes in the presence or absence of antimicrobials provides an alternative approach to traditional culture for faster AST. In this review, we summarize recent developments in rapid AST methods through metabolic profiling of bacteria under antibiotic treatment. Full article

A rapid and reliable diagnostic for tuberculosis, including the detection of both rifampicin (RIF) and isoniazid (INH) resistance, is essential for appropriate patient care. Nucleic acid amplification tests are a fast alternative to methods based on Mycobacterium tuberculosis complex (MTB) cultures. Thus, the performance of the MDR/MTB ELITe MGB^® Kit on the ELITe InGenius^® platform was retrospectively evaluated for MTB detection on pulmonary and extra-pulmonary samples and for RIF/INH resistance detection on MTB strains. The sensitivity and specificity of the kit for MTB detection compared to the MTB culture were 80.0% and 100.0%, respectively. For the antimicrobial susceptibility prediction, the agreement with phenotypic antimicrobial susceptibility testing (AST) was 92.0%. For RIF, the sensitivity was 100.0% and the specificity was 95.5%. For INH, the sensitivity and specificity were 75.0% and 100.0%, respectively. A single RIF false-positive result was obtained for a strain with a low level of RIF resistance that was not detected by phenotypic AST, but carrying a rpoB L452P mutation. INH false-negative results (3) were due to mutations on the katG gene that were not probed by the test. Overall, the MTB/MDR ELITe MGB^® Kit presents a strong performance for MTB detection and for the detection of both RIF and INH resistance, with an easy integration in laboratory workflow thanks to its fully automatized system. Full article