The incidence of candidemia caused by
and
is constantly increasing and is accompanied by the rising use of the few available antifungals. The widespread use of echinocandins and azoles for the ...treatment of invasive candidemia has enhanced the development of antifungal resistance, resulting in an increasing health care problem. Hence, the rapid detection of resistant strains is required. This study aimed to evaluate the detection of
and
strains resistant to caspofungin by the matrix-assisted laser desorption ionization Biotyper antibiotic susceptibility test rapid assay (MBT ASTRA). This novel semiquantitative technique facilitates the detection of caspofungin-resistant strains within 6 h. MBT ASTRA results were compared to the data obtained by the use of Clinical and Laboratory Standards Institute (CLSI) guidelines. Clinical isolates of
(
= 58) and
(
= 57) were analyzed by MBT ASTRA and the CLSI microdilution method. Antifungal susceptibility testing against caspofungin by the CLSI microdilution method classified the
isolates into 36 susceptible and 22 resistant strains and the
isolates into 5 susceptible, 33 resistant, and 19 intermediate strains. For
, the comparison of MBT ASTRA and the CLSI method revealed an excellent categorical agreement of 100%. A sensitivity and a specificity between MBT ASTRA and the CLSI microdilution method of 94% and 80%, respectively, were detected for
strains, based on categorical agreement. In conclusion, the results obtained by MBT ASTRA indicate that this is a very promising approach for the rapid detection of
isolates resistant to caspofungin.
MALDI‐TOF MS profiling for microorganism detection has already been demonstrated in the 1990s, but has evolved to the first‐line identification method in many laboratories just during the past five ...years. While this application of MALDI‐TOF MS has proven its broad applicability, accuracy, robustness, and cost‐effectiveness it is of particular interest to expand the capabilities of the mass spectrometric platform. Resistance detection is the most desirable further application of MALDI‐TOF MS in microbiology, but maybe also the most challenging. Different approaches have been published regarding diverse antibiotic drugs and distinct microorganism classes. The current review shall give an overview about the developments of the recent years and their potential to get transformed in clinical useful assays in the future.
Species identification and drug susceptibility testing (DST) of mycobacteria are important yet complex processes traditionally reserved for reference laboratories. Recent technical improvements in ...matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has started to facilitate routine mycobacterial identifications in clinical laboratories. In this paper, we investigate the possibility of performing phenotypic MALDI-based DST in mycobacteriology using the recently described MALDI Biotyper antibiotic susceptibility test rapid assay (MBT-ASTRA). We randomly selected 72 clinical Mycobacterium tuberculosis and nontuberculous mycobacterial (NTM) strains, subjected them to MBT-ASTRA methodology, and compared its results to current gold-standard methods. Drug susceptibility was tested for rifampin, isoniazid, linezolid, and ethambutol (M. tuberculosis, n = 39), and clarithromycin and rifabutin (NTM, n = 33). Combined species identification was performed using the Biotyper Mycobacteria Library 4.0. Mycobacterium-specific MBT-ASTRA parameters were derived (calculation window, m/z 5,000 to 13,000, area under the curve AUC of >0.015, relative growth RG of <0.5; see the text for details). Using these settings, MBT-ASTRA analyses returned 175/177 M. tuberculosis and 65/66 NTM drug resistance profiles which corresponded to standard testing results. Turnaround times were not significantly different in M. tuberculosis testing, but the MBT-ASTRA method delivered on average a week faster than routine DST in NTM. Databases searches returned 90.4% correct species-level identifications, which increased to 98.6% when score thresholds were lowered to 1.65. In conclusion, the MBT-ASTRA technology holds promise to facilitate and fasten mycobacterial DST and to combine it directly with high-confidence species-level identifications. Given the ease of interpretation, its application in NTM typing might be the first in finding its way to current diagnostic workflows. However, further validations and automation are required before routine implementation can be envisioned.
The development of new techniques for the detection of carbapenemase activity is of great importance since the increased incident of resistance against carbapenems represents a serious threat to ...global public health. In this context, the matrix-assisted laser desorption/ionization approach already demonstrated to be a reliable tool for rapid carbapenemase detection. As a newly developed test, there is still a lack of in-depth analysis of its robustness and possible wider application. The main goal of this study was to evaluate the potential for using the design MBT STAR-Carba assay as the pre-characterization method for Enterobacterales and P. aeruginosa strains in terms of the produced classes of carbapenemases using modified procedure parameters-various suspension densities and incubation times. Moreover, its usefulness for the in-depth analysis and characterization of metallo-β-lactamases (MBL) was tested by applying inhibition assays. In this study, the designed assay proved to be a sensitive tool for the detection of carbapenemase hydrolytic activity, which can be successfully used to partially classify the class of carbapenemase present. Additionally, the use of defined high concentration suspensions would allow to shorten the incubation time to 1 minute for certain strains. Considering that the assay was also suitable to investigate the effect of different inhibitors on the MBL activity, it demonstrates far higher discriminatory potential than only a rapid routine carbapenemase detection tool and could be used as a susceptibility assay.
Antibiotic resistant bacteria are a growing concern worldwide. Extended-spectrum β-lactamases (ESBL) represent the most common resistance mechanism of Gram-negative bacteria against β-lactams, ...underlining the need for adequate diagnostic methods that provide reliable information in the shortest time possible. AmpC, a less prevalent but increasingly relevant class of β-lactamases, pose an additional challenge as their detection is complex. Here, we present an ESBL and AmpC screening panel employing the MALDI-TOF MS-based direct-on-target microdroplet growth assay (DOT-MGA).
Four reference strains recommended by the European Committee on Antimicrobial Susceptibility Testing (EUCAST) were used to develop the panel, which was further validated on 50 clinical
isolates resistant to third generation cephalosporins. The panel relies on the synergistic effect between ESBL and/or AmpC β-lactamase inhibitors and cephalosporins, which indicates β-lactamase production. Microdroplets containing the tested microorganism, cephalosporins in different concentrations and inhibitors were pipetted onto an MBT Biotarget and incubated for 3 or 4 h at 35 ± 1°C. Afterward, the liquid medium was removed and the material adhered to the spots was analyzed by MALDI-TOF MS. Synergy was detected by determining and comparing the minimum inhibitory concentrations of the tested cephalosporins with and without β-lactamase inhibitors. Data were interpreted following a diagnostic algorithm proposed by EUCAST in order to establish a final diagnosis. In comparison, PCR, broth microdilution (BMD) and combination disk tests (CDT) were performed.
Compared to the PCR results, the following positive and negative percent agreement values (PPA/NPA) were obtained for each resistance mechanism: ESBL, 94.44/100%; AmpC, 94.44/93.75% and ESBL+AmpC, 100/100%. These results, obtained after 4 h of incubation, were comparable with those of BMD and showed a higher accuracy than CDT.
We propose a novel phenotypic method for detection of ESBL and AmpC β-lactamases in
that provides reliable results in a short time, representing a promising alternative to the diagnostic techniques currently available. This easy-to-perform approach has potential for being implemented in routine laboratories, contributing to the further diversification of mass spectrometry technology into other fields such as antibiotic resistance testing.
was first reported in an ear swab from Japan in 2009; it then promptly spread over five continents and turned into a global nosocomial problem. The main challenges faced by many researchers are the
...-identification by conventional methods in clinical laboratories and failure in treatment. About 90% of
strains are intrinsically resistant to fluconazole (FLU), and it is developing resistance to multiple classes of available antifungals. Echinocandins are the most potent class of antifungals against
; however, reduced susceptibility to one or many echinocandin drugs has been recently observed. Thus, the main issues addressed in this paper are the fast and accurate identification of
derived from Sabouraud dextrose agar and blood culture bottles as well as the rapid antifungal susceptibility test by MALDI-TOF MS. This study successfully identified all isolates of
(
= 50) by MALDI-TOF MS, with an average log score of ≥ 2. An accuracy of 100% was found on both agar plate and blood culture bottles. MALDI Biotyper antibiotic susceptibility test-rapid assay (MBT ASTRA) was used for rapid antifungal susceptibility testing (AFST). A comparison between MBT ASTRA and the Clinical and Laboratory Standards Institute guidelines (CLSI) detected a sensitivity and specificity of 100% and 98% for anidulafungin, and 100% and 95.5% for micafungin, respectively. A categorical agreement of 98% and 96% was calculated for the two methods. For caspofungin, sensitivity and specificity of 100 and 73% were found, respectively, with a categorical agreement of 82%. MBT ASTRA has the great potential to detect
isolates non-susceptible against echinocandin antifungals within 6 h, which makes it a promising candidate for AFST in clinical laboratories in the future.
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