Quality Control Isolates Research Articles

A New Method for Determination of Rifampicin and Isoniazid Resistance in Mycobacterium tuberculosis complex Isolates: Capillary Tube Method

Tuberculosis continues to be an important public health problem worldwide. Culture methods are still considered the gold standard in the diagnosis of tuberculosis and the determination of drug resistance. The most important limitation of these methods is their long turnaround time. Commercial culture systems developed to shorten the duration are emerging as an economic problem, especially for developing countries. Therefore, cheap, fast, easy to apply and objectively evaluable tests are needed. In this study, in addition to culture-based methods for determining RIF and INH resistance in Mycobacterium tuberculosis complex isolates, it was aimed to develop the capillary tube method to accelerate the evaluation process. The study included 27 RIF-resistant, 36 RIF -sensitive, 30 INH-resistant, and 33 INH-sensitive isolates obtained from the mycobacteriology laboratory culture collection, for which susceptibility testing to firstline drugs were previously performed using the BACTEC MGIT 960 system (BD, USA) and were stored. H37Rv standard strain and an external quality control strain (IDT3) with known RIF and INH resistance were used as quality control isolates in the study. As a new testing method, the capillary tube method for detecting rifampicin and isoniazid resistance was compared to the standard BACTEC MGIT 960 system. In the determination of RIF and INH resistance, the sensitivity of the capillary tube method compared to the reference method was determined as 85% and 80%, respectively; however, the specificity values (25% and 45.5%, respectively) for both drugs were found to be low in the studies. The time to detect resistance with the capillary tube method varied between 4-9 days. Capillary tube method, which was developed especially for the rapid identification and treatment of multidrug-resistant isolates, is promising in that it detects resistant strains in a short time with a relatively high sensitivity, although its specificity is very low. It is thought that it would be beneficial to continue the study with a larger number of samples and even improve the method with studies conducted directly from clinical samples.

The Influence of Medium Composition on EUCAST and Etest Antifungal Susceptibility Testing.

There is an ongoing effort to optimize and revise antifungal susceptibility testing (AFST) methods due to the rising number of fungal infections and drug-resistant fungi. The rising antifungal resistance within Candida and Aspergillus species, which are common contributors to invasive fungal infections (IFIs), is a cause for concern, prompting an expanding integration of in vitro AFST to guide clinical decisions. To improve the relevance of in vitro AFST results to therapy outcomes, influential factors should be taken into account. The tested medium is one of several factors that could affect the results of AFST. The present study evaluated the effect of two complex media (Sabouraud dextrose and Columbia) versus the standard defined medium (RPMI 1640) on the AFST results of amphotericin B, posaconazole, and voriconazole against Candida spp. and Aspergillus spp. representatives, utilizing the European Committee on Antimicrobial Susceptibility Testing (EUCAST) and the Etest methods. Overall, Candida species exhibited higher variability in minimum inhibitory concentration (MIC) across different media (more than three log2 dilutions) comparing to Aspergillus spp., while quality control isolates showed consistency regardless of tested media, antifungals, and methods. When comparing tested methods, MIC variation was mostly detected using EUCAST than it was using Etest.

Employing Flow Cytometry to Extracellular Vesicles Sample Microvolume Analysis and Quality Control.

Extracellular Vesicles (EVs), membrane vesicles released by all cells, are emerging mediators of cell-cell communication. By carrying biomolecules from tissues to biofluids, EVs have attracted attention as non-invasive sources of clinical biomarkers in liquid biopsies. EVs-based liquid biopsies usually require EVs isolation before content analysis, which frequently increases sample volume requirements. We here present a Flow Cytometry (FC) strategy that does not require isolation or concentration of EVs prior to staining. By doing so, it enables population analysis of EVs in samples characterized by challenging small volumes, while reducing overall sample processing time. To illustrate its application, we performed longitudinal non-lethal population analysis of EVs in mouse plasma and in single-animal collections of murine vitreous humor. By quantifying the proportion of vesicular particles in purified and non-purified biological samples, this method also serves as a precious tool to quality control isolates of EVs purified by different protocols. Our FC strategy has an unexplored clinical potential to analyze EVs in biofluids with intrinsically limited volumes and to multiply the number of different analytes in EVs that can be studied from a single collection of biofluid.

The accuracy of four commercial broth microdilution tests in the determination of the minimum inhibitory concentration of colistin

Colistin is considered as one of the last-resort antibiotics and reliable antimicrobial susceptibility testing is therefore crucial. The reference standard for AST according to EUCAST and CLSI is broth microdilution (BMD). However, BMD is labor intensive to perform. Commercial antimicrobial susceptibility tests derived from BMD method are available. We investigated the performance of four different commercial tests: Sensititre™, SensiTest™ Colistin, Micronaut MIC Strip Colistin and UMIC Colistin using 70 clinical isolates (half of them was deemed by VITEK2 as resistant), including isolates from cystic fibrosis patients and mcr-1 bearing isolates. We used two reference standards: BMD and composite MIC as determined by all four tests. Sensititre™ had essential agreement (EA, defined as minimum inhibitory concentration within ± 1 dilution) of 87% and 89% compared to BMD and composite reference standard, respectively. For SensiTest™, the EA’s were 93% and 90%. For UMIC, 87% and 90%, and for Micronaut, 83% and 84%. All four tests demonstrated categorical agreement (CA) above 90%. CA for SensiTest™ and Micronaut was both 96%, UMIC 94%, and Sensititre™ 93%. All tests were reproducible as tested in two quality control isolates. In conclusion, in clinical isolates from a large referral center, the four commercial tests for determination of colistin minimum inhibitory concentrations showed acceptable performance.

Stop Waiting for Tomorrow: Early Disk Diffusion—An Accurate Method for Antimicrobial Susceptibility Testing With Reduced Turnaround Time

Abstract Background Disk diffusion is a slow but reliable reference method for measuring antimicrobial susceptibility. Our objective was to improve the turnaround time for this method by reducing the culture incubation period prior to disk diffusion testing (referred to as early disk diffusion [eDD] testing). Methods Clinical isolates (n = 13) and quality control (QC) strains (n = 8) of bacteria, including 6 Staphylococcus aureus, 3 Enterococcus faecium, 3 Enterococcus faecalis, 3 Escherichia coli, 3 Pseudomonas aeruginosa, 2 Klebsiella pneumoniae, and 1 Enterobacter cloacae, were inoculated on blood agar by quadrant streaking and were incubated at 35○C for 6 hours (eDD6), 10 hours (eDD10), or 24 hours (standard [sDD]) before disk diffusion testing was set up in accordance with CLSI guidelines using Mueller Hinton Agar (Hardy Diagnostics) and clinically appropriate antimicrobial agents (total: 24). Experiments were performed sequentially in triplicate with zones measured by two independent readers. Results Examination of 6-hour blood agar plates showed limited growth in the first 1 to 2 quadrants while 10-hour growth plates had 4-quadrant growth and individual colonies in most cases. Despite limited growth on 6-hour plates, there were adequate bacteria to produce a 0.5 McFarland standard for disk diffusion testing for all 126 eDD replicates. Results were evaluable for 1,206 of 1,206 (100%) of eDD and 603 of 603 (100%) of standard disk diffusion (sDD). A comparison of early vs standard disk diffusion showed that eDD6 had 3 of 154 (1.9%) very major errors and 6 of 449 (1.3%) major errors, whereas eDD10 had no very major errors and 3 of 449 (0.7%) minor errors. Very major errors in eDD6 included a 1-mm difference between eDD6 and sDD for cefoxitin inhibition of S aureus as well as 4- and 6-mm differences between eDD6 replicates and sDD for nitrofurantoin inhibition of E cloacae. There was similar categorical agreement of eDD6 (96.7%) and eDD10 (96.7%) with sDD. Overall, there was a very good correlation of eDD6 (r2 = 0.98) and eDD10 (r2 = 0.99) with sDD. When grouping results by bacterial species, there was no more than 1-mm difference in median antimicrobial inhibition between eDD6, eDD10, and sDD. Of note, 5 of 7 bacterial species had the same median zone size when comparing eDD6 or eDD10 with sDD. There was also very little difference between early and standard methods when examining bacteria-drug combinations, with a similar zone size (difference in mode <2 mm) in 67 of 69 (97%) eDD6 and 66 of 69 (96%) eDD10 compared to sDD. Test results for QC isolates agreed with the CLSI recommended ranges in 97 of 99 (98%) eDD6, 97 of 99 (98%) eDD10, and 96 of 99 (97%) sDD. Conclusion Early disk diffusion testing is a simple and accurate method of antimicrobial susceptibility testing that can reduce time to results by as much as 18 hours with no additional cost.

Evaluation of the performance of MALDI-TOF MS and DNA sequence analysis in the identification of mycobacteria species

Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry is an alternative way of identifying mycobacteria via the analysis of biomolecules. It is being increasingly used in routine microbiology practice since it permits early, rapid, and cost-effective identification of pathogens of clinical importance. In this study, we aimed to evaluate the efficacy of phenotypic identification of mycobacteria by the MALDI-TOF MS MBT Mycobacteria Library (ML) 4.0 (Bruker, Daltonics) compared to standard sequence analysis. A total of 155 Mycobacterium clinical and external quality control isolates, comprising nontuberculous mycobacteria (NTM) (n = 95) and the Mycobacterium tuberculosis complex (MTC) (n = 60), were included in the study. Identification by MBT ML4.0 was correctly performed in 100% of MTC and in 91% of NTM isolates. All of the MTC isolates were correctly differentiated from NTM isolates. Based on our results, MBT ML4.0 may be used reliably to identify both NTM and MTC.

1144. Modern Problem, Medieval Cure-Resistant Aeromonas in Medicinal Leeches

BackgroundMedicinal leeches are used primarily in plastic and reconstructive surgery when venous congestion threatens tissue viability. The associated infection risk ranges from 4.1 to 20%. Prophylactic antimicrobials such as fluoroquinolones (FQ) or trimethoprim-sulfamethoxazole (SXT) are recommended and target commonly isolated pathogen and gut symbiont, Aeromonas. However, resistance to these agents has been reported and detected in leeches, including at hospital systems across Canada that acquire their stock from the same supplier. Our objective was to describe the local epidemiology of leech-related Aeromonas resistant to one or more commonly used prophylactic agents, and determine if such resistance originates from the common supplier.MethodsSix hospital systems across Canada using leech therapy, purchased from the same supplier, were surveyed. A 5-year retrospective review of all antimicrobial resistant leech-related Aeromonas, derived from clinical, leech, and tank fluid specimens was performed. All Aeromonas resistant to either FQ or SXT were included, and retained frozen isolates from each system were analysed by pulse-field gel electrophoresis (PFGE) using a published Aeromonas protocol.ResultsAll six hospital systems reported leech-related Aeromonas resistant to one or more antimicrobials, totalling 15 isolates. Three systems only reported data from the last year. Four systems used FQ and two used SXT as prophylaxis. Fifteen of 15 were either FQ resistant or intermediate, and four of 15 were SXT resistant. Three of 10 isolates tested for ceftriaxone (CRO) susceptibility were resistant. Five of 15 of the isolates were resistant to two or more agents. Of the two leech quality control isolates, 2/2 were FQ resistant and 1/2 was FQ, SXT and CRO resistant. Only three isolates, each from a different, geographically distinct hospital system, had been retained. PFGE analysis indicated 2/3 are closely related (Figure 1).ConclusionOur preliminary investigation suggests that the presence of FQ and SXT resistance in leech-related Aeromonas might be more common than previously suspected, and that such resistance might originate from a common source. A broader study of the molecular epidemiology of leech-related Aeromonas is warranted. Disclosures All authors: No reported disclosures.

Direct identification of clinical pathogens from liquid culture media by MALDI-TOF MS analysis

ObjectivesWe propose using MALDI-TOF MS as a tool for identifying microorganisms directly from liquid cultures after enrichment of the clinical sample in the media, to obtain a rapid microbiological diagnosis and an adequate administration of the antibiotic therapy in a clinical setting. MethodsTo evaluate this approach, a series of quality control isolates were grown in thioglycollate (TG) broth and brain heart infusion (BHI) broth and extracted under four different protocols before finally being identified by MALDI-TOF MS. After establishing the best extraction protocol, we validated the method in a total of 300 liquid cultures (150 in TG broth and 150 in BHI broth) of different types of clinical samples obtained from two tertiary Spanish hospitals. ResultsThe initial evaluation showed that the extraction protocol including a 5 minute sonication step yielded 100% valid identifications, with an average score value of 2.305. In the clinical validation of the procedure, 98% of the microorganisms identified from the TG broth were correctly identified relative to 97% of those identified from the BHI broth. In 24% of the samples analysed, growth by direct sowing was only successful in the liquid medium, and no growth was observed in the direct solid agar cultures. ConclusionsUse of MALDI-TOF MS plus the sonication-based extraction method enabled direct and accurate identification of microorganisms in liquid culture media in 15 minutes, in contrast to the 24 hours of subculture required for conventional identification, allowing the administration of a targeted antimicrobial therapy.

Multilaboratory Evaluation of In Vitro Antifungal Susceptibility Testing of Dermatophytes for ME1111.

ME1111 is a novel small molecule antifungal agent under development for the topical treatment of onychomycosis. Standardization of the susceptibility testing method for this candidate antifungal is needed. Toward this end, 8 independent laboratories determined the interlaboratory reproducibility of ME1111 susceptibility testing. In addition, we subsequently identified 2 strains as quality control (QC) isolates for the method. In the reproducibility study, 5 blinded clinical strains each of Trichophyton rubrum, Trichophyton mentagrophytes, and Epidermophyton floccosum were tested, while the QC study tested 6 blinded T. rubrum or T. mentagrophytes ATCC strains. Testing was performed in frozen microtiter panels according to the Clinical and Laboratory Standards Institute (CLSI) M38-A2 methodology. In the reproducibility study, 9 of 15 clinical strains showed interlaboratory agreement of >90% at the 80% inhibition endpoint, with a range of agreement of 76.2% to 100%. In the QC study, 4 of the 6 ATCC strains showed interlaboratory agreement of >90%. ME1111 demonstrated excellent interlaboratory agreement when tested against dermatophytes. Based on this data, the CLSI Subcommittee on Antifungal Susceptibility Tests approved the susceptibility testing of ME1111 against dermatophytes according to M38-A2 methodology, which stipulates RPMI 1640 as the test medium, an inoculum size of 1 to 3 × 10(3) CFU/ml, and an incubation time and temperature of 96 h at 35°C. The MIC endpoint should be 80% inhibition compared with the growth control. T. rubrum ATCC MYA-4438 and T. mentagrophytes ATCC 28185 were selected as QC isolates, with an acceptable range of 0.12 to 1 μg/ml for the two strains.

Interlaboratory Study of Quality Control Isolates for a Broth Microdilution Method (Modified CLSI M38-A) for Testing Susceptibilities of Dermatophytes to Antifungals

The Clinical and Laboratory Standards Institute (CLSI; formerly National Committee for Clinical Laboratory Standards, or NCCLS) M38-A standard for the susceptibility testing of filamentous fungi does not specifically address the testing of dermatophytes. In 2003, a multicenter study investigated the reproducibility of the microdilution method developed at the Center for Medical Mycology, Cleveland, Ohio, for testing the susceptibility of dermatophytes. Data from that study supported the introduction of this method for testing dermatophytes in the future version of the CLSI M38-A standard. In order for the method to be accepted by CLSI, appropriate quality control isolates needed to be identified. To that end, an interlaboratory study, involving the original six laboratories plus two additional sites, was conducted to evaluate potential candidates for quality control isolates. These candidate strains included five Trichophyton rubrum strains known to have elevated MICs to terbinafine and five Trichophyton mentagrophytes strains. Antifungal agents tested included ciclopirox, fluconazole, griseofulvin, itraconazole, posaconazole, terbinafine, and voriconazole. Based on the data generated, two quality control isolates, one T. rubrum isolate and one T. mentagrophytes isolate, were identified and submitted to the American Type Culture Collection (ATCC) for inclusion as reference strains. Ranges encompassing 95.2 to 97.9% of all data points for all seven drugs were established.

Multisite reproducibility of MIC results by the Sensititre ® YeaStone Colorimetric Antifungal Susceptibility Panel

Reproducibility of MIC results between laboratories, a major performance criterion used for evaluation of any susceptibility test method, was determined at three test sites using the Sensititre ® YeastOne Antifungal Panel, which incorporates Alamar Blue TM as a colorimetric indicator. MICs of five antifungals were determined using a set of 10 isolates of Candida species. Each isolate was tested a total of nine times against each antifungal agent in each of the three laboratories. A total of 1350 MICs were evaluated. MICs were read visually after incubation at 35°C for 24 and 48 h. Overall, 99 to 100% of MIC values were encompassed by a range defined by the modal MIC ± 1 dilution for each antifungal agent tested at both 24 h and 48 h. Replicate testing of the quality control isolates recommended by the National Committee for Clinical Laboratory Standards demonstrated excellent agreement between results obtained with the Sensititre ® YeastOne panel and the MIC reference range for each antifungal agent. These studies demonstrated that the Sensititre ® YeastOne Antifungal Panel may be used to generate MIC values for at least five different antifungal agents with a high degree of intra- and interlaboratory reproducibility.

Multisite reproducibility of colorimetric broth microdilution method for antifungal susceptibility testing of yeast isolates

MICs of fluconazole and amphotericin B were determined independently for 100 coded yeast isolates by each of six laboratories to determine reproducibility of results by using a colorimetric oxidation-reduction-based broth microdilution test. In addition, each site tested five quality control isolates on at least four different occasions during the study. Results agreed within a three-dilution range (mode +/- 1 log2 dilution) for 96.2% of fluconazole tests and 92.7% of amphotericin B tests. Agreement among tests with the quality control isolates was 99.4% with fluconazole and 98.6% with amphotericin B. These results indicate that the colorimetric microdilution method is reproducible among laboratories.

Identification of veterinary pathogens by use of commercial identification systems and new trends in antimicrobial susceptibility testing of veterinary pathogens

Veterinary diagnostic microbiology is a unique specialty within microbiology. Although isolation and identification techniques are similar to those used for human pathogens, many veterinary pathogens require unique cultivation or identification procedures. Commercial identification systems provide rapid, accurate identification of human pathogens. However, the accuracy of these systems with veterinary pathogens varies widely depending on the bacterial species and the host animal from which it was isolated. Increased numbers of veterinary strains or species in the data bases of the various systems would improve their accuracy. Current procedures and interpretive criteria used for antimicrobial susceptibility testing of veterinary pathogens are based on guidelines used for human pathogens. The validity of these guidelines for use with veterinary pathogens has not been established. As with fastidious human pathogens, standardized methodologies and quality control isolates are needed for tests of organisms such as Actinobacillus pleuropneumoniae and Haemophilus somnus. Furthermore, interpretive criteria for veterinary antimicrobial agents based on the MIC for veterinary pathogens, the pharmacokinetics of the antimicrobial agent in the host animal, and in vivo efficacy of the antimicrobial agent are needed. This article reviews both the commercial identification systems evaluated with veterinary pathogens and current methods for performing and interpreting antimicrobial susceptibility tests with veterinary pathogens. Recommendations for future improvements in both areas are discussed.

Selection of candidate quality control isolates and tentative quality control ranges for in vitro susceptibility testing of yeast isolates by National Committee for Clinical Laboratory Standards proposed standard methods

The National Committee for Clinical Laboratory Standards has developed a proposed standard method for in vitro antifungal susceptibility testing of yeast isolates (National Committee for Clinical Laboratory Standards, document M27-P, 1992). In order for antifungal testing by the M27-P method to be accepted, reliable quality control (QC) performance criteria must be developed. In the present study, five laboratories tested 10 candidate QC strains 20 times each against three antifungal agents: amphotericin B, fluconazole, and 5-fluorocytosine. All sites conformed to the M27-P standards and used a common lot of tube dilution reagents and RPMI 1640 broth medium. Overall, 98% of MIC results with amphotericin B, 95% with fluconazole, and 92% with 5-fluorocytosine fell within the desired 3-log2 dilution range (mode +/- 1 log2 dilution). Excellent performance with all three antifungal agents was observed for six strains: Candida albicans ATCC 90028, Candida parapsilosis ATCC 90018, C. parapsilosis ATCC 22019, Candida krusei ATCC 6258, Candida tropicalis ATCC 750, and Saccharomyces cerevisiae ATCC 9763. With these strains, 3-log2 dilution ranges encompassing 94 to 100% of MICs for all three drugs were established. Additional studies with multiple lots of RPMI 1640 test medium will be required to establish definitive QC ranges.

Identification of veterinary pathogens by use of commercial identification systems and new trends in antimicrobial susceptibility testing of veterinary pathogens.

Veterinary diagnostic microbiology is a unique specialty within microbiology. Although isolation and identification techniques are similar to those used for human pathogens, many veterinary pathogens require unique cultivation or identification procedures. Commercial identification systems provide rapid, accurate identification of human pathogens. However, the accuracy of these systems with veterinary pathogens varies widely depending on the bacterial species and the host animal from which it was isolated. Increased numbers of veterinary strains or species in the data bases of the various systems would improve their accuracy. Current procedures and interpretive criteria used for antimicrobial susceptibility testing of veterinary pathogens are based on guidelines used for human pathogens. The validity of these guidelines for use with veterinary pathogens has not been established. As with fastidious human pathogens, standardized methodologies and quality control isolates are needed for tests of organisms such as Actinobacillus pleuropneumoniae and Haemophilus somnus. Furthermore, interpretive criteria for veterinary antimicrobial agents based on the MIC for veterinary pathogens, the pharmacokinetics of the antimicrobial agent in the host animal, and in vivo efficacy of the antimicrobial agent are needed. This article reviews both the commercial identification systems evaluated with veterinary pathogens and current methods for performing and interpreting antimicrobial susceptibility tests with veterinary pathogens. Recommendations for future improvements in both areas are discussed.