Direct Antibiotic Susceptibility Research Articles

Evaluation of three protocols for direct susceptibility testing for Gram-negative rods from flagged positive blood culture bottles.

Bloodstream infections are associated with high mortality that can be reduced by targeted antibiotic therapy in the early stages of infection. Direct antibiotic susceptibility testing (AST) from flagged positive blood cultures may facilitate the administration of early effective antimicrobials much before the routine AST. Clinical and Laboratory Standards Institute-recommended direct AST can be performed with a limited number of antibiotic disks only. On the other hand, using an automated system for direct AST will not only allow effective laboratory workflow with reduced turnaround time but also provide the minimum inhibitory concentration values of tested antibiotics. However, using expensive automated systems for direct AST may not be feasible for resource-limited laboratories. Therefore, in this study, we aimed to evaluate the CLSI-recommended method and two other direct AST protocols (one with an automated system and the other with disk diffusion) for Gram-negative rods from flagged positive blood cultures.

Evaluation of Chromogenic Agar Media for Isolation, Identification and Direct Antibiotic Susceptibility Testing of Uropathogens

Evaluation of Chromogenic Agar Media for Isolation, Identification and Direct Antibiotic Susceptibility Testing of Uropathogens

Rapid diagnosis of bacteria and determination of sensitivity in blood culture

Amaç: Kan dolaşımı enfeksiyonu olan hastalarda; etkenin kısa sürede tanımlanması ve uygun antimikrobiyal tedavi uygulanması, morbidite ve mortalitenin azaltılması bakımından oldukça önemlidir. Bu çalışmada, kan kültüründen doğrudan tanımlama ve antibiyotik duyarlılık testlerinin yapılması için geliştirilen yeni bir yöntemin değerlendirilmesi amaçlanmıştır. Gereç ve Yöntem: BacT/Alert 3D sisteminde pozitif sinyal veren kan kültürü örneklerinden yıkama ve santrifüj işlemleri ile bakteriyel çökelti elde edildi. Bu çökeltiden Vitek MS kullanılarak tanımlama yapıldı, ardından VITEK 2 otomatize sisteminde doğrudan antibiyotik duyarlılık testi çalışıldı. Sonuçlar standart yöntem ile karşılaştırıldı. Bulgular: Tanımlama işlemi 80 kan kültürü örneğinde gerçekleştirildi. Doğrudan tanımlama işleminde 73 örnek tanımlandı ve bunlardan 72’si (%90) standart yöntemle uyumlu olarak sonuçlandı. Doğrudan antibiyotik duyarlılık testlerinin %97,9 oranında uyumlu olduğu saptandı. Değerlendirilen 635 antibiyotik duyarlılık sonucu içinde; 10’unda büyük hata, 3’ünde küçük hata olduğu görüldü. Sonuç: Kan kültürü örneklerinden çalışmada uygulanan prosedürler kullanılarak 24 saat içinde, maliyetli reaktifler ya da uzun işlem süresine gereksinim olmadan, standart uygulama sonuçlarına benzer bir şekilde tanımlama ve antibiyotik duyarlılık sonucu elde edilebileceği görülmüştür

Rapid diagnosis and reduced workload for urinary tract infection using flowcytometry combined with direct antibiotic susceptibility testing.

We evaluated if flowcytometry, using Sysmex UF-5000, could improve diagnosis of urinary tract infections by rapid identification of culture negative and contaminated samples prior to culture plating, thus reducing culture plating workload and response time. We also evaluated if it is possible to reduce the response time for antibiotic susceptibility profiles using the bacteria information flag on Sysmex UF-5000 to differentiate between Gram positive and negative bacteria, followed by direct Antibiotic Susceptibility Testing (dAST) on the positive urine samples. One thousand urine samples were analyzed for bacteria, white blood cells and squamous cells by flowcytometry before culture plating. Results from flowcytometric analysis at different cut-off values were compared to results of culture plating. We evaluated dAST on 100 urine samples that were analyzed as positive by flowcytometry, containing either Gram positive or Gram negative bacteria. Using a cut-off value with bacterial count ≥100.000/mL and WBCs ≥10/μL, flowcytometry predicted 42,1% of samples with non-significant growth. We found that most contaminated samples contain few squamous cells. For 52/56 positive samples containing Gram negative bacteria dAST was identical to routine testing. Overall, there was concordance in 555/560 tested antibiotic combinations. Flowcytometry offers advantages for diagnosis of urinary tract infections. Screening for negative urine samples on the day of arrival reduces culture plating and workload, and results in shorter response time for the negative samples. The bacteria information flag predicts positive samples containing Gram negative bacteria for dAST with high accuracy, thus Antibiotic Susceptibility Profile can be reported the day after arrival. For the positive samples containing Gram negative bacteria the concordance was very good between dAST and Antibiotic Susceptibility Testing in routine. For positive samples containing Gram positive bacteria the results were not convincing. We did not find any correlation between epithelial cells and contamination.

Direct microfluidic antibiotic resistance testing in urine with smartphone capture: significant variation in sample matrix interference between individual human urine samples.

Rapid and portable direct tests for antibiotic resistance in human clinical samples such as urine could reduce misuse of precious antimicrobials, by allowing treatment decisions to be informed by microfluidic diagnostic tests. We demonstrate that the variable composition of human urine can significantly affect the antibiotic minimum inhibitory concentration (MIC) measured using microfluidic devices. The urine sample matrix interference was not observed in pooled normal urine, emphasising the critical importance of assessing matrix interference with a wide range of individual urine samples, rather than a few standardised or pooled controls. Both dilution into assay medium and inclusion of buffer could reduce the matrix interference, but dilution may affect analytical sensitivity by increasing the minimum bacterial cell density needed in a sample for growth to be detected, especially for miniaturised devices that test small sample volumes. We conclude it is vital to fully assess and optimise novel analytical microbiology tools using multiple individual urine samples, otherwise the high variation in matrix interference will compromise the clinical performance of these rapid diagnostics that are urgently needed to tackle the global threat of antimicrobial resistance.

Direct Detection of Methicillin Resistant Staphylococci: Comparison of Phenotypic Methods with Multiplex PCR and Direct Susceptibility Testing

Bloodstream infections are augean diseases characterized by a high morbidity and mortality, related with the lag in administration of the first adequate anti-infectious agent. Clinical and epidemiological data guide the clinicians towards empirical anti-infectious treatments whose effectualness remains questionable especially in the present day milieu of multidrug-resistant organisms. Early microbiological evidence of the causative agent is hierophantic of antimicrobial stewardship. We evaluated three rapid methods for the direct identification of S. aureus from 720 positive blood cultures in comparison to Vitek 2 automated microbial identification system. Early distinction S. aureus from CoNS was attempted with Direct Tube Coagulase. Multiplex PCR assay was used to separate MRSA from MSSA by the presence of mecA gene. Direct antibiotic susceptibility (DST) from positive blood culture bottles was performed in an endeavour to reduce time and compared to the routine Vitek 2 compact antimicrobial susceptibility. For Direct tube coagulase at 4 hrs of incubation sensitivity was 79.6% while specificity was 100% however, on overnight incubation sensitivity increased to 97.9% with 100% specificity. The positive predictive value and negative predictive values were 100% and 96.9% respectively when compared to Vitek 2 identification of staphylococcal species. By DST Categorical agreement of 95% was seen for coagulase negative staphylococci microorganism-antibiotic combinations. Categorical agreement of 89.7% was seen for S. aureus microorganism-antibiotic combinations. Direct multiplex PCR testing did not misidentify any S. aureus isolate compared to Vitek 2. In 12 methicillin resistant CoNS isolates mecA gene was not detected by PCR. In total 13(3.5%) strains of staphylococci identified by DST as methicillin resistant were not identified by PCR analysis. Each of the tests has positive qualities and all may have a place in a Gram positive cocci algorithm for testing blood cultures depending on the laboratory setting, workload volume and staffing. However, rapid detection methods are a pressing priority.

Direct antibiotic susceptibility testing of blood cultures of gram-negative bacilli using the Drug Susceptibility Testing Microfluidic (DSTM) device

Proper treatment of bloodstream infections requires rapid, early determination of appropriate antibiotic agents, emphasizing the need for more rapid drug susceptibility testing. The Drug Susceptibility Testing Microfluidic (DSTM) device represents a novel method in which a small amount of bacterial suspension is injected into the microchip-like device and cultured for 3 h. However, it remains unknown whether the DSTM method can directly determine antibiotic susceptibilities from positive blood cultures. Here, we developed a new approach to directly assess drug susceptibility, using the DSTM method for positive blood cultures. We compare the utility and accuracy of DSTM with those of conventional susceptibility testing methods. Fifty positive blood cultures identified as gram-negative bacilli were used herein. The outcomes of drug susceptibility and resistance assays for positive blood cultures were compared to those of conventional susceptibility testing methods to evaluate their utility and accuracy. Method agreement rates between DSTM and standard methods often exceed 90%, suggesting a high positive correlation with conventional methods. Furthermore, our results show that a combination of multiple drugs in the DSTM device helps identify extended-spectrum β-lactamase (ESBL)- and AmpC-β-lactamase (AmpC-)-producing microorganisms. In conclusion, DSTM method enables effective drug susceptibility and resistance screening within 3 h from positive blood cultures and is suitable for the rapid and personalized determination of the antimicrobial regimen.

Evaluation of direct drug susceptibility testing of blood culture isolates comparing it with conventional disk diffusion testing.

To evaluate a direct antibiotic susceptibility testing method for blood culture.. The cross-sectional comparative study was conducted at the Armed Forces Institute of Pathology, Rawalpindi, Pakistan, from December 2016 to October 2017. Direct antimicrobial susceptibility testing was performed from positive blood culture bottles. Bacterial identification was done by using API 10S. Different antimicrobial panels were employed for Gram-negative rods (GNRs), gram-positive cocci (like suspected Staphylococci and Enterococci). Results were compared with conventional disk diffusion testing and very major, major and minor errors were calculated. Result agreement and kappa coefficient scores were generated for categorical agreement. SPSS 24 was used for data analysis. Of the 101 bacterial isolates, 82(81.2%) were Gram negative rods and 19(18.8%) were Grampositive cocci. Among 781 bacteria-antibiotic comparisons, the number of very major errors was 3(0.4%), major errors were 7(0.9%) and minor errors were 12(1.5%), while, 759(97.2%) comparisons yielded the same results. The kappa coefficient was 0.946, showing almost perfect agreement. Direct identification of Gram negative rods was successful in 53(64.6%) cases. Direct susceptibility testing of blood culture produced reliable results for majority of the antimicrobials.

Comparision of Direct Antibiotic Susceptibility Testing with Standard Testing in Blood Culture

Comparision of Direct Antibiotic Susceptibility Testing with Standard Testing in Blood Culture

Simple Sample Preparation Method for Direct Microbial Identification and Susceptibility Testing From Positive Blood Cultures.

Rapid identification and determination of the antibiotic susceptibility profiles of the infectious agents in patients with bloodstream infections are critical steps in choosing an effective targeted antibiotic for treatment. However, there has been minimal effort focused on developing combined methods for the simultaneous direct identification and antibiotic susceptibility determination of bacteria in positive blood cultures. In this study, we constructed a lysis-centrifugation-wash procedure to prepare a bacterial pellet from positive blood cultures, which can be used directly for identification by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS) and antibiotic susceptibility testing by the Vitek 2 system. The method was evaluated using a total of 129 clinical bacteria-positive blood cultures. The whole sample preparation process could be completed in <15 min. The correct rate of direct MALDI-TOF MS identification was 96.49% for gram-negative bacteria and 97.22% for gram-positive bacteria. Vitek 2 antimicrobial susceptibility testing of gram-negative bacteria showed an agreement rate of antimicrobial categories of 96.89% with a minor error, major error, and very major error rate of 2.63, 0.24, and 0.24%, respectively. Category agreement of antimicrobials against gram-positive bacteria was 92.81%, with a minor error, major error, and very major error rate of 4.51, 1.22, and 1.46%, respectively. These results indicated that our direct antibiotic susceptibility analysis method worked well compared to the conventional culture-dependent laboratory method. Overall, this fast, easy, and accurate method can facilitate the direct identification and antibiotic susceptibility testing of bacteria in positive blood cultures.

Droplet-based non-faradaic impedance sensors for assessment of susceptibility of Escherichia coli to ampicillin in 60 min.

Direct antibiotic susceptibility tests (AST) are essential for rapid detection of bacterial infection and administration of appropriate antibiotics. Conventional AST systems are usually slow as they rely on cell growth for an indirect assessment of antibiotics' effectiveness. Therefore, a faster method is desirable, especially for emergency cases. In this work, we studied the performance of label-free, droplet-based impedance sensors for rapid characterization of the effects of ampicillin (Amp) on Escherichia coli. Ampicillin damages cell wall integrity and makes cells permeable (leaky). The leakage results in significant increase of the electrical conductance measured directly by the microfabricated sensing unit. We studied the conductance signal as a function of both antibiotic treatment time and dosage and demonstrated susceptibility testing within 60min. These findings demonstrate the potential of droplet-based electrical chips for the realization of electrical antibiotic susceptibility testing (e-AST) for early-stage diagnostic/treatment, and consequently, preventing antibiotic misuse/overuse.

Diagnostic Value of Direct Antibiotic Susceptibility Test for Faster BacterialSusceptibility Reporting in Bacteremia

Background: Rapid and accurate information on susceptibility of bacteria causing bacteraemia is very helpful in sepsis management. Blood culture is the gold standard for bacteraemia diagnosis. Standard antibiotic susceptibility testing needs at least three days for completion while direct method can give the result a day earlier. The aim of this study was to investigate the diagnostic value of direct antibiotic susceptibility testing in blood culture.Methods: Bloods from positive BACTEC bottles which met inclusion and exclusion criteria were put into sterile tubes and centrifuged. The pellets were then used to make 0.5 McFarland bacterial suspensions and directly used for antibiotic susceptibility testing. Interpretations of direct method were compared to standard method to count sensitivity, specificity, sensitive predictive value, resistant predictive value, accuracy, and kappa value.Results: From 58 samples (containing 22 gram negative, 36 gram positive bacteria), there were 309 total antibiotic susceptibility tests. Direct method showed sensitivity, specificity, sensitive predictive value, resistant predictive value, accuracy, and kappa value of 89.3%, 92.9%, 93.8%, 87.8%, 86.4%, and 0.82, respectively.Conclusion: Direct antibiotic susceptibility testing has a good agreement with the standard method so it can aid faster antibiotic susceptibility reporting in bacteraemia (Sains Medika, 4(2):174-181).

Tentative Direct Antimicrobial Susceptibility Testing in Urine

Purpose Tentative direct disk susceptibilities in bacteriuria compared to standard techniques were reevaluated. Materials and Methods A sterile cotton swab was saturated with urine and uniformly streaked over the surface of a Mueller-Hinton agar plate. Susceptibility disks were added within 5 minutes and the zone of inhibition around the antibiotic disk was read after 16 to 18 hours of incubation. Results The tentative direct susceptibilities showed an accuracy of 98.2 percent compared to standard techniques in 2,906 positive pure cultures. The false-positive rate was 0.53 percent and the false-negative rate was 0.14 percent. Conclusions Results of tentative direct antibiotic susceptibility in bacteriuria are comparable to those of standard techniques and are obtained 24 hours sooner.

New aid to management of septicaemia: rapid direct antibiotic susceptibility testing.

A rapid and reliable method of performing direct antibiotic susceptibility tests on positive blood cultures has been developed. A result is now available five hours after laboratory confirmation of septicaemia. An evaluation involving 55 blood-culture isolates demonstrated an over-all correlation of 97.8% when results obtained by the rapid direct Autobac l method were compared to those obtained by definitive testing procedures. The method is particularly applicable to isolates of Staphyloccus aureus, Pseudomonas aeruginosa, and the Enterobacteriaceae. It is especially significant because of the recent emergence of multiple antibiotic resistance amongst hospital strains of these bacteria with many isolates showing resistance to antibiotics frequently used in the empirical treatment of septicaemia. The rapid availability of a reliable antibiogram is important in allowing early appropriate chemotherapy and, hence, in the reduction of septicaemia-associated morbidity and mortality, and the length of stay in hospital.