Rapid Susceptibility Testing Method Research Articles

Rapid Identification of Escherichia coli Colistin-Resistant Strains by MALDI-TOF Mass Spectrometry.

Colistin resistance is one of the major threats for global public health, requiring reliable and rapid susceptibility testing methods. The aim of this study was the evaluation of a MALDI-TOF mass spectrometry (MS) peak-based assay to distinguish colistin resistant (colR) from susceptible (colS) Escherichia coli strains. To this end, a classifying algorithm model (CAM) was developed, testing three different algorithms: Genetic Algorithm (GA), Supervised Neural Network (SNN) and Quick Classifier (QC). Among them, the SNN- and GA-based CAMs showed the best performances: recognition capability (RC) of 100% each one, and cross validation (CV) of 97.62% and 100%, respectively. Even if both algorithms shared similar RC and CV values, the SNN-based CAM was the best performing one, correctly identifying 67/71 (94.4%) of the E. coli strains collected: in point of fact, it correctly identified the greatest number of colS strains (42/43; 97.7%), despite its lower ability in identifying the colR strains (15/18; 83.3%). In conclusion, although broth microdilution remains the gold standard method for testing colistin susceptibility, the CAM represents a useful tool to rapidly screen colR and colS strains in clinical practice.

In-house pre-prepared broth microdilution plates: A simple, cheap and pragmatic approach for susceptibility testing of colistin

Background: With the upsurge in multi-drug resistant Gram-negative bacilli, it is imperative to optimize and standardize the antibiotic susceptibility testing (AST) methods for polymyxins (colistin & polymyxin B). The long overdue confusion regarding the susceptibility testing of colistin was put to rest in 2016 when the CLSI-EUCAST joint Polymyxin Breakpoint Working Group recommended broth microdilution (BMD) in polystyrene plates as the reference method for AST. Though several commercially-available BMD plates have been assessed by EUCAST, the high cost of such commercial plates during contemporary times hinder their use in routine microbiological laboratories especially in low and middle income countries. Thus, a suitable alternative to commercial BMD plates should be sought for to ensure a cost-effective, rapid and reliable reporting of AST of colistin. Methods and materials: With the aim of reducing this redundancy in the workflow, we designed an experiment to charge several microtiter plates with the drug dilution and the media on day 0, storing them frozen at −200C and then retrieving them sequentially for AST (henceforth called pre-prepared frozen BMD plates (PFBP). The PFBP were charged with same representative organisms on every 2–4 days for two months to check for the reliability of AST reporting. Essential agreement (EA), categorical agreement (CA), very major error (VME) and major errors (ME) were computed in accordance with ISO standard 20776-2. For validation, the criteria of EA3 90%, CA 3 90%, VME £1.5% AND ME£3% was used. Pearson correlation and Cohen's kappa (k) score was calculated using GraphPad (San Diego, CA, USA). Results: For colistin, EA and CA of PFBP was 97.5% and 98.3%, respectively. For polymyxin B, EA and CA of PFBP was 99.1% and 90%. Further, the performance of PFBP for colistin susceptibility testing of A. baumannii in the present study (EA=97.5%, CA=98.3%) was superior to five commercial plates available and evaluated by EUCAST. Conclusion: Based on these observations, we propose that pre-prepared frozen BMD plates can be incorporated in routine workflow of microbiological laboratory as reliable, cheap and rapid method for susceptibility testing of colistin.

137. Impact of Rapid Susceptibility Testing on Outcomes in Patients with Bacteremia

BackgroundEarly organism identification via rapid diagnostics has been shown to reduce time to effective antimicrobial therapy and improve patient outcomes in patients with bacteremia, but antimicrobial susceptibility testing is still required to optimize therapy. The objective of this study was to determine the impact of an institution-specific rapid susceptibility testing method on outcomes in patients with bacteremia.MethodsThis was a retrospective pre- and post-intervention study of 100 adult patients with bacteremia. Patients were excluded if they had polymicrobial infection, fungemia, blood cultures collected at outside hospitals, or if they expired prior to susceptibility results. Patients were identified through a report containing positive blood cultures from October 2017 to February 2018 (pre-intervention [PrI]) and October 2018 to February 2019 (post-intervention [PoI]). The primary endpoint was the rate of clinical failure (a composite of 28-day mortality or bacteremia persisting greater than 6 days). Secondary endpoints included microbiologic outcomes, time to effective and optimal therapy, length of stay (LOS) and therapy adjustments.ResultsBaseline characteristics were similar between groups; a third of the patients were immunosuppressed (Table 1). The most common sources of infection were urinary and intra-abdominal, and the most common organisms identified were E.coli and Klebsiella spp. No significant difference in the rate of clinical failure was identified between PrI and PoI (24% vs. 18%, P = 0.6242) (Table 2). In the PoI, the time to identification, susceptibility results, and effective therapy was significantly shorter with similar time to optimal therapy and LOS. In the PoI, antimicrobial stewardship program (ASP) interventions were made significantly sooner after susceptibility results.ConclusionIn this small, retrospective, single-center study, the implementation of a rapid susceptibility testing method was associated with reduced time to susceptibility results and more rapid interventions by the ASP, but no difference in the rate of clinical failure or time to optimal therapy was identified. Disclosures All authors: No reported disclosures.

Rapid susceptibility testing of multi-drug resistant Escherichia coli and Klebsiella by glucose metabolization monitoring.

Background The increasing number of multi-drug resistant (MDR) bacteria provides enormous challenges for choosing an appropriate antibiotic therapy in the early phase of sepsis. While bacterial identification has been greatly accelerated by the introduction of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), the antibiotic susceptibility testing (AST) remains time-consuming. Here, we present a rapid susceptibility testing method for testing Gram-negative bacteria, exemplarily validated for Escherichia coli and Klebsiella spp. Methods Gram-negative isolates (E. coli and Klebsiella spp.) were either taken as single colonies from agar plates (n=136) or directly extracted and identified from positive blood cultures (n=42) using MALDI-TOF MS. Bacteria were incubated in glucose-supplemented Luria broths (LBs) each containing one antibiotic (ceftazidime, piperacillin, imipenem and ciprofloxacin), routinely used to classify Gram-negative bacteria in Germany. To determine susceptibility the dynamics of glucose utilization in bacterial suspensions were quantitatively measured in the presence or absence of antibiotics designated liquid-AST (L-AST). Results The L-AST can be run on clinical-chemistry analyzers and integrated into laboratory routines. It yields critical resistance information within 90-150 min downstream of a MS-based identification. The results showed a high concordance with routine susceptibility testing, with less than 1% very major errors (VME) and 3.51% major errors (ME) for 178 assessed isolates. Analysis of turnaround time (TAT) for 42 clinical samples indicated that L-AST results could be obtained 34 h earlier than the routine results. Conclusions As exemplified for E. coli and Klebsiella spp., L-AST provides substantial acceleration of susceptibility testing following MALDI-TOF MS identification. The assay is a simple and low-cost method that can be integrated into clinical laboratory to allow for 24/7 AST. This approach could improve antibiotic therapy.

A rapid method for post-antibiotic bacterial susceptibility testing.

Antibiotic susceptibility testing is often performed to determine the most effective antibiotic treatment for a bacterial infection, or perhaps to determine if a particular strain of bacteria is becoming drug resistant. Such tests, and others used to determine efficacy of candidate antibiotics during the drug discovery process, have resulted in a demand for more rapid susceptibility testing methods. Here, we have developed a susceptibility test that utilizes chemiluminescent determination of ATP release from bacteria and the overall optical density (OD600) of the bacterial solution. Bacteria release ATP during a growth phase or when they are lysed in the presence of an effective antibiotic. Because optical density increases during growth phase, but does not change during bacterial lysing, an increase in the ATP:optical density ratio after the bacteria have reached the log phase of growth (which is steady) would indicate antibiotic efficacy. Specifically, after allowing a kanamycin-resistant strain of Escherichia coli (E.coli) to pass through the growth phase and reach steady state, the addition of levofloxacin, an antibiotic to which E. coli is susceptible, resulted in a significant increase in the ATP:OD600 ratio in comparison to the use of kanamycin alone (1.80 +/- 0.50 vs. 1.12 +/- 0.28). This difference could be measured 20 minutes after the addition of the antibiotic, to which the bacteria are susceptible, to the bacterial sample. Furthermore, this method also proved useful with gram positive bacteria, as the addition of kanamycin to a chloramphenicol-resistant strain of Bacillus subtilis (B. subtilis) resulted in an ATP:OD600 ratio of 2.14 +/- 0.26 in comparison to 0.62 +/- 0.05 for bacteria not subjected to the antibiotic to which the bacteria are susceptible. Collectively, these results suggest that measurement of the ATP:OD600 ratio may provide a susceptibility test for antibiotic efficacy that is more rapid and quantitative than currently accepted techniques.

New method for rapid Susceptibility Testing on blood culture with HB&L system: preliminary data

Blood culture, although represents the gold standard in detecting the ethiological agent of sepsis, is rather rarely required in relation to the real diagnostic importance. The result of this test depends in fact on many factors (sample volume, time of collection, accuracy, antibiotic therapy, contamination, number of drawings, drawing site, interpretation difficulties, etc.) that are often considered by many clinicians so limited as to doubt about their actual value. The disadvantages are therefore represented by the lack of standardization but also by the low sensitivity and above all by the technical times too long for the clinical needs. Blood culture begins with the drawing of samples from the “septic” patient followed incubation of the bottles in automatic thermostated systems. In case of positive result (36 hours), the culture is Gram stained and streaked on solid media in order to obtain isolated colonies for the identification and the susceptibility testing (48 hours from positive result). The long time required for pathogen identification and susceptibility testing involves empirical broad spectrum antibiotic therapy that can promote the increase of bacterial resistance but also patient management costs. A clinically useful report should be available on short notice in order to guide the clinician to choose the most appropriate antibiotic. The microbiologist has therefore the hard work of reviewing the organization and the management of the procedures.We have therefore started to consider the possibility of treating the blood as an biological liquid in order to quickly determine the susceptibility of bacteria to antibiotics.

Direct fluconazole susceptibility testing of positive Candida blood cultures by flow cytometry

The standard methods for yeast susceptibility testing require 24-48 h of incubation. As there has been an increase in incidence of non-albicans Candida species, the clinician is very often wary of initiating therapy with fluconazole until a final susceptibility report is generated, especially when treating very sick patients. A rapid reliable susceptibility testing method would enable the clinician to prescribe fluconazole, thus avoiding more toxic or expensive therapy. To determine the feasibility of direct susceptibility testing of Candida species to fluconazole by a rapid flow cytometric method, 50 Candida strains were seeded into blood culture bottles and were tested for susceptibility to fluconazole directly from the bottles after their being flagged as positive by the blood culture instrument. Minimal inhibitory concentration (MIC) determined by fluorescent flow cytometry (FACS) showed excellent agreement to that determined by macrodilution. Following the seeding experiments, 30 true patient specimens were tested directly from positive blood cultures, and MIC determined by both methods showed excellent agreement. Antifungal susceptibility testing by FACS directly from positive blood culture bottles is a reliable, rapid method for susceptibility testing of Candida to fluconazole. The method allows same-day results, does not require subculture to agar media, and can greatly assist in the selection of appropriate antifungal therapy.

Use of high inoculum for early metabolic signalling and rapid susceptibility testing of Aspergillus species

To develop and evaluate a new method for rapid susceptibility testing of Aspergillus spp. based on early metabolic signalling of high-inoculum biomass. Susceptibility to amphotericin B and voriconazole was studied in 39 clinical isolates of Aspergillus spp. (16 Aspergillus fumigatus, 11 Aspergillus flavus, 12 Aspergillus terreus). At 6 or 8 h after inoculation for A. fumigatus and A. flavus, and at 8 or 12 h after inoculation for A. terreus, 100 microg/mL of the tetrazolium salt XTT and 25 microM menadione were added and absorbance measured at 450 nm after 2 h of incubation at 37 degrees C. Inocula used were 10(6) conidia/mL for A. fumigatus and A. terreus and 10(5) conidia/mL for A. flavus, as lower inocula exhibited very low metabolic activity at these time points. Data were analysed with the sigmoid E(max) model and compared with visual (lowest drug concentration showing no growth) and spectrophotometric MIC determination at 48 h (CLSI M38-A method). The E(max) model described well the concentration-effect relationship for early metabolic activity and 48 h fungal biomass (median r(2): 0.97 and 0.93, respectively). Use of the model allowed characterization and quantification of species- and drug-related differences in pharmacological inhibition of early metabolic activity as well as calculation of appropriate cutoff levels for MIC determination with the XTT assay. Using these cutoff levels, for A. fumigatus and A. flavus at both time points (6 and 8 h) and for A. terreus at 12 h, the agreement (+/- one dilution) of the XTT assay with the CLSI method was 91-100% and its reproducibility was 97-100%. This newly developed high-inoculum-based method provides rapid and reproducible MIC determinations for Aspergillus spp.

Rapid mycobacteria drug susceptibility testing using Gel Microdrop (GMD) Growth Assay and flow cytometry

Control of multi-drug-resistant tuberculosis has been hampered by the lack of simple, rapid and sensitive methods for assessing bacterial growth and antimicrobial susceptibility. Due to the increasing incidence and high frequency of mutations, it is unlikely that culture methods will disappear in the foreseeable future. Therefore, the need to modernize methods for rapid detection of viable clinical isolates, at a minimum as a gold standard, will persist. Previously, we confirmed the feasibility of using the Gel Microdrop (GMD) Growth Assay for identifying sub-populations of resistant Mycobacteria by testing different laboratory strains. Briefly, this assay format relies on encapsulating single bacterium in agarose microspheres and identifying clonogenic growth using flow cytometry and fluorescent staining. In this study, we modified the GMD Growth Assay to make it suitable for clinical applications. We demonstrated the effectiveness and safety of this novel approach for detecting drug susceptibility in clinically relevant laboratory strains as well as clinical isolates of Mycobacterium tuberculosis. Correlation between results using the GMD Growth Assay format and results using two well characterized methods (Broth Microdilution MIC and BACTEC 460TB) was 87.5% and 90%, respectively. However, due to the inherent sensitivity of flow cytometry and the ability to detect small (<1%) sub-populations of resistant mycobacteria, the GMD Growth Assay identified more cases of drug resistance. Using 4 clinically relevant mycobacterial strains, we assessed susceptibility to primary anti-tuberculosis drugs using both the Broth Microdilution MIC method and the GMD Growth Assay. We performed 24 tests on isoniazid-resistant BCG, Mycobacterium tuberculosis H 37 Ra and Mycobacterium avium strains. The Broth Microdilution MIC method identified 7 cases (29.1%) of resistance to INH and EMB compared to the GMD Growth Assay which identified resistance in 10 cases (41.6%); in 3 cases (12.5%), resistance to INH and EMB was detected only with the GMD Growth Assay. In addition, using 20 Mycobacterium tuberculosis clinical isolates, we compared results using BACTEC 460TB method performed by collaborators and the GMD Growth Assay. Eight of 20 (40%) clinical isolates, which were not identified as drug-resistant using the conventional BACTEC 460TB method, were resistant to 1, 2, or 3 different concentrations of drugs using the GMD Growth Assay (13 cases of 140 experiments). In one case (isolate 1879), resistance to 10.0 μg/ml of STR detected using BACTEC 460TB method was not confirmed by the GMD Growth Assay. Thus, the overall agreement between these methods was 90% (14 discrepant results of 140 experiments). These data demonstrate that the GMD Growth Assay is an accurate and sensitive method for rapid susceptibility testing of Mycobacterium tuberculosis for use in clinical reference laboratory settings.

Evaluation of a new method for rapid drug susceptibility testing of Mycobacterium avium complex isolates by using the mycobacteria growth indicator tube.

The reliability of the Mycobacteria Growth Indicator Tube (MGIT [BBL]) for rapid drug susceptibility testing of Mycobacterium avium complex (MAC) isolates was evaluated. MICs of amikacin, clarithromycin, clofazimine, ethambutol, and rifabutin were determined by the MGIT system for 16 MAC strains. The results were compared with those obtained by the BACTEC broth macrodilution method. The turnaround times were 6 to 8 days (median, 7 days) for the MGIT and 5 to 7 days (median, 6 days) for the BACTEC system. Agreements with BACTEC system-determined MICs, within +/-1 log2 dilution, were 100, 100, 88, 63, and 44% for amikacin, clofazimine, rifabutin, clarithromycin, and ethambutol, respectively. Within +/-2 log2 dilutions, agreement with BACTEC system-determined MICs increased to 100% for all the tested drugs. In addition, if MGIT-determined MICs were evaluated according to the thresholds adopted for the interpretation of BACTEC system-determined ones, ethambutol was the only drug for which susceptible strains were frequently misclassified as resistant. It is concluded that the MGIT system is a promising, nonradiometric alternative to the BACTEC method for rapid susceptibility testing of MAC isolates; however, additional studies are required to confirm our results and to determine the optimal criteria for the interpretation of ethambutol MICs.

Rapid drug susceptibility testing of Mycobacterium tuberculosis using conventional solid media.

Radiometric methods for M. tuberculosis drug susceptibility testing yield much faster results than standard techniques; however, these methods require sophisticated equipment and are expensive. We investigated a rapid drug susceptibility testing method for isoniazid, rifampin, ethambutol, streptomycin and pyrazinamide in specimens from 197 patients with pulmonary tuberculosis using a simplified agar-dilution method. Middlebrook 7H11 agar solid medium and microcolony detection were used to test sputum from 64 smear-positive, and from 70 culture-positive but smear-negative patients. Culture-positive material from bronchoscopy, surgical biopsy, pleural fluid or gastric fluid of 63 patients was tested. In 64 smear-positive patients, the median time for final susceptibility results was 11 days (95% confidence interval (95% CI) 10-12 days) compared to 62 days (95% CI 56-66 days) with the standard method. In 133 smear-negative patients, results were available after a median of 35 days (95% CI 32-40 days) in contrast to 72 days (95% CI 62-83 days) with the standard method, regardless of whether or not sputum or other materials were used for primary culture. The rapid method detected all cases of single-drug resistance (n = 20) and multidrug resistance (n = 14) within 13 days (95% CI 9-17 days) in smear-positive patients (n = 8), or within 38 days (95% CI 35-48 days) in smear-negative patients (n = 26). Only one discrepancy was encountered in 985 resistance tests. Moreover, contamination was not observed. Our rapid susceptibility testing method for M. tuberculosis on Middlebrook 7H11 agar is fast, practical and inexpensive. It provides an alternative when more sophisticated techniques are not available or affordable.

A rapid susceptibility testing method for yeasts to 5‐fluorocytosine

A rapid method of susceptibility testing of yeasts to 5-fluorocytosine is demonstrated. The method is based on photometric determination of the optical density of yeast suspensions before and after incubation for 4 h in the presence of the antifungal. The method was evaluated with 50 Candida strains.