scholarly journals Epidemiological cutoff values for a 96-well broth microdilution plate for high-throughput research antibiotic susceptibility testing of M. tuberculosis

2021 ◽  
Author(s):  
◽  
Philip W Fowler
Keyword(s):  
Genetic Variants ◽  
Susceptibility Testing ◽  
Drug Susceptibility ◽  
Drug Susceptibility Testing ◽  
Broth Microdilution ◽  
Wild Type ◽  
Genetic Sequencing ◽  
Indicator Tube ◽  
Cutoff Values ◽  
Type Population

AbstractDrug susceptibility testing of M. tuberculosis is rooted in a binary susceptible/resistant paradigm. There are considerable advantages in measuring the minimum inhibitory concentrations (MICs) of a panel of drugs for an isolate, including quantifying the magnitude of effect conferred by genetic variants and being able to identify isolates with elevated MICs that can still be treated with standard therapy. It is necessary, however, to measure the epidemiological cutoff values (ECOFF/ECVs) to permit comparison with qualitative data. Here we present ECOFF/ECVs for 13 anti-TB compounds, including bedaquiline and delamanid, derived from 20,637 clinical isolates collected by 14 laboratories based in 11 countries on five continents. Each isolate was incubated for 14 days on a dry 96-well broth microdilution plate and then read. Resistance to the majority of the drugs due to prior exposure is expected and the MIC distributions for many of the compounds are complex and therefore a phenotypically wild-type population could not be defined. Since a majority of samples also underwent genetic sequencing, we defined a genotypically wild-type population and measured the MIC of the 99th percentile by direct measurement and via fitting a Gaussian using interval regression. The proposed ECOFF/ECV values were then validated by comparing to the MIC distributions of high-confidence genetic variants that confer resistance and to qualitative drug susceptibility tests obtained via Mycobacterial Growth Indicator Tube and the Microscopic-Observation Drug-Susceptibility assay.

scholarly journals Antifungal Susceptibility Testing of Malassezia spp. with an Optimized Colorimetric Broth Microdilution Method

2017 ◽  
Vol 55 (6) ◽  
pp. 1883-1893 ◽  
Author(s):  
Cheryl Leong ◽  
Antonino Buttafuoco ◽  
Martin Glatz ◽  
Philipp P. Bosshard
Keyword(s):  
Susceptibility Testing ◽  
Skin Diseases ◽  
Antifungal Susceptibility ◽  
Drug Susceptibility ◽  
Drug Susceptibility Testing ◽  
Broth Microdilution ◽  
Content Type ◽  
Microdilution Method ◽  
Broth Microdilution Method ◽  
Colorimetric Indicator

ABSTRACTMalasseziais a genus of lipid-dependent yeasts. It is associated with common skin diseases such as pityriasis versicolor and atopic dermatitis and can cause systemic infections in immunocompromised individuals. Owing to the slow growth and lipid requirements of these fastidious yeasts, convenient and reliable antifungal drug susceptibility testing assays forMalasseziaspp. are not widely available. Therefore, we optimized a broth microdilution assay for the testing ofMalasseziathat is based on the CLSI and EUCAST assays forCandidaand other yeasts. The addition of ingredients such as lipids and esculin provided a broth medium formulation that enabled the growth of allMalasseziaspp. and could be read, with the colorimetric indicator resazurin, by visual and fluorescence readings. We tested the susceptibility of 52 strains of 13Malasseziaspecies to 11 commonly used antifungals. MIC values determined by visual readings were in good agreement with MIC values determined by fluorescence readings. The lowest MICs were found for the azoles itraconazole, posaconazole, and voriconazole, with MIC90values of 0.03 to 1.0 μg/ml, 0.06 to 0.5 μg/ml, and 0.03 to 2.0 μg/ml, respectively. AllMalasseziaspp. were resistant to echinocandins and griseofulvin. SomeMalasseziaspp. also showed high MIC values for ketoconazole, which is the most widely recommended topical antifungal to treatMalasseziaskin infections. In summary, our assay enables the fast and reliable susceptibility testing ofMalasseziaspp. with a large panel of different antifungals.

scholarly journals A Multilaboratory, Multicountry Study To Determine MIC Quality Control Ranges for Phenotypic Drug Susceptibility Testing of Selected First-Line Antituberculosis Drugs, Second-Line Injectables, Fluoroquinolones, Clofazimine, and Linezolid

2016 ◽  
Vol 54 (12) ◽  
pp. 2963-2968 ◽  
Author(s):  
Koné Kaniga ◽  
Daniela M. Cirillo ◽  
Sven Hoffner ◽  
Nazir A. Ismail ◽  
Devinder Kaur ◽  
...  
Keyword(s):  
Quality Control ◽  
Reference Strain ◽  
Susceptibility Testing ◽  
Drug Susceptibility ◽  
Drug Susceptibility Testing ◽  
Second Line ◽  
Broth Microdilution ◽  
First Line ◽  
Antituberculosis Drugs ◽  
Content Type

Our objective was to establish reference MIC quality control (QC) ranges for drug susceptibility testing of antimycobacterials, including first-line agents, second-line injectables, fluoroquinolones, and World Health Organization category 5 drugs for multidrug-resistant tuberculosis using a 7H9 broth microdilution MIC method. A tier-2 reproducibility study was conducted in eight participating laboratories using Clinical Laboratory and Standards Institute (CLSI) guidelines. Three lots of custom-made frozen 96-well polystyrene microtiter plates were used and prepared with 2× prediluted drugs in 7H9 broth-oleic acid albumin dextrose catalase. The QC reference strain wasMycobacterium tuberculosisH37Rv. MIC frequency, mode, and geometric mean were calculated for each drug. QC ranges were derived based on predefined, strict CLSI criteria. Any data lying outside CLSI criteria resulted in exclusion of the entire laboratory data set. Data from one laboratory were excluded due to higher MIC values than other laboratories. QC ranges were established for 11 drugs: isoniazid (0.03 to 0.12 μg/ml), rifampin (0.03 to 0.25 μg/ml), ethambutol (0.25 to 2 μg/ml), levofloxacin (0.12 to 1 μg/ml), moxifloxacin (0.06 to 0.5 μg/ml), ofloxacin (0.25 to 2 μg/ml), amikacin (0.25 to 2 μg/ml), kanamycin (0.25 to 2 μg/ml), capreomycin (0.5 to 4 μg/ml), linezolid (0.25 to 2 μg/ml), and clofazimine (0.03 to 0.25 μg/ml). QC ranges could not be established for nicotinamide (pyrazinamide surrogate), prothionamide, or ethionamide, which were assay nonperformers. Using strict CLSI criteria, QC ranges against theM. tuberculosisH37Rv reference strain were established for the majority of commonly used antituberculosis drugs, with a convenient 7H9 broth microdilution MIC method suitable for use in resource-limited settings.

scholarly journals Evaluation of a New Method for Rapid Drug Susceptibility Testing of Mycobacterium avium Complex Isolates by Using the Mycobacteria Growth Indicator Tube

1998 ◽  
Vol 36 (1) ◽  
pp. 64-67 ◽  
Author(s):  
Claudio Piersimoni ◽  
Domenico Nista ◽  
Stefano Bornigia ◽  
Giuseppina De Sio
Keyword(s):  
Mycobacterium Avium ◽  
Mycobacterium Avium Complex ◽  
Susceptibility Testing ◽  
Drug Susceptibility ◽  
Drug Susceptibility Testing ◽  
New Method ◽  
Indicator Tube ◽  
Broth Macrodilution ◽  
Bactec System ◽  
Growth Indicator

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.

scholarly journals Validation of Bedaquiline Phenotypic Drug Susceptibility Testing Methods and Breakpoints: a Multilaboratory, Multicountry Study

2020 ◽  
Vol 58 (4) ◽  
Author(s):  
Koné Kaniga ◽  
Akio Aono ◽  
Emanuele Borroni ◽  
Daniela Maria Cirillo ◽  
Christel Desmaretz ◽  
...  
Keyword(s):  
Susceptibility Testing ◽  
Improve Patient Care ◽  
Drug Susceptibility ◽  
Drug Susceptibility Testing ◽  
Error Rates ◽  
World Health ◽  
Health Concern ◽  
Content Type ◽  
Indicator Tube ◽  
Health Organization

ABSTRACT Drug-resistant tuberculosis persists as a major public health concern. Alongside efficacious treatments, validated and standardized drug susceptibility testing (DST) is required to improve patient care. This multicountry, multilaboratory external quality assessment (EQA) study aimed to validate the sensitivity, specificity, and reproducibility of provisional bedaquiline MIC breakpoints and World Health Organization interim critical concentrations (CCs) for categorizing clinical Mycobacterium tuberculosis isolates as susceptible/resistant to the drug. Three methods were used: Middlebrook 7H11 agar proportion (AP) assay, broth microdilution (BMD) assay, and mycobacterial growth indicator tube (MGIT) assay. Each of the five laboratories tested the 40-isolate (20 unique isolates, duplicated) EQA panel at three time points. The study validated the sensitivity and specificity of a bedaquiline MIC susceptibility breakpoint of 0.12 μg/ml for the BMD method and WHO interim CCs of 1 μg/ml for MGIT and 0.25 μg/ml for the 7H11 AP methods. Categorical agreements between observed and expected results and sensitivities/specificities for correctly identifying an isolate as susceptible/resistant were highest at the 0.25, 0.12, and 1 μg/ml bedaquiline concentrations for the AP method, BMD (frozen or dry plates), and MGIT960, respectively. At these concentrations, the very major error rates for erroneously categorizing an isolate as susceptible when it was resistant were the lowest and within CLSI guidelines. The most highly reproducible bedaquiline DST methods were MGIT960 and BMD using dry plates. These findings validate the use of standardized DST methodologies and interpretative criteria to facilitate routine phenotypic bedaquiline DST and to monitor the emergence of bedaquiline resistance.

scholarly journals Occurrence ofrpoBMutations in Isoniazid-Resistant but Rifampin-Susceptible Mycobacterium tuberculosis Isolates from Germany

2013 ◽  
Vol 58 (1) ◽  
pp. 590-592 ◽  
Author(s):  
Sönke Andres ◽  
Doris Hillemann ◽  
Sabine Rüsch-Gerdes ◽  
Elvira Richter
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Susceptibility Testing ◽  
Drug Susceptibility ◽  
Drug Susceptibility Testing ◽  
Content Type ◽  
Indicator Tube ◽  
Lowenstein Jensen ◽  
Proportion Method ◽  
Rifampin Resistance ◽  
Growth Indicator

ABSTRACTFour out of 143 phenotypically isoniazid-resistant but rifampin-susceptibleMycobacterium tuberculosisstrains that were isolated from patients in Germany in 2011 had mutations in the rifampin resistance-determining region ofrpoB. After performing drug susceptibility testing (DST) with two methods, the proportion method on Löwenstein-Jensen medium and using the Bactec 960 Mycobacteria Growth Indicator Tube system, we conclude that the two methods are equally reliable for phenotypic DST and MIC determination.

scholarly journals Direct detection of resistance to fluoroquinolones/SLIDs in sputum specimen by GenoType MTBDRsl v.2.0 assay A study from Eastern Uttar Pradesh, India

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Kamal Singh ◽  
Richa Kumari ◽  
Smita Gupta ◽  
Rajneesh Tripathi ◽  
Anjali Srivastava ◽  
...  
Keyword(s):  
Susceptibility Testing ◽  
Direct Detection ◽  
Uttar Pradesh ◽  
Drug Susceptibility ◽  
Drug Susceptibility Testing ◽  
High Rate ◽  
World Health ◽  
Second Line ◽  
Indicator Tube ◽  
Health Organization

Abstract Background According to World Health Organization (WHO), drug-resistant tuberculosis (DR-TB) is a major contributor to antimicrobial resistance globally and continues to be a public health threat. Annually, about half a million people fall ill with DR-TB globally. The gradual increase in resistance to fluoroquinolones (FQs) and second-line injectable drugs (SLIDs), poses a serious threat to effective TB control and adequate patient management. Therefore, WHO suggests the use of GenoType MTBDRsl v.2.0 assay for detection of multiple mutations associated with FQs and SLIDs. Hence, the study was conducted to determine the prevalence of resistance to FQs and SLIDs by comparing direct GenoType MTBDRsl v.2.0 assay with phenotypic drug susceptibility testing (DST). Methods The study was conducted on 1320 smear positive sputum samples from a total of 2536 RR-TB, confirmed by GeneXpert MTB/RIF. The smear positive specimens were decontaminated, and DNA extraction was performed. Furthermore, the extracted DNA was used for GenoType MTBDRsl v.2.0 assay. While 20% of the decontaminated specimens were inoculated in Mycobacterium growth indicator tube (MGIT) for drug susceptibility testing (DST). Results Out of 1320 smear positive sputum samples, 1178 were identified as Mycobacterium tuberculosis complex (MTBC) and remaining were negative by GenoType MTBDRsl v.2.0 assay. Of the 1178 MTBC positive, 26.6% were sensitive to both FQs and SLIDs, whereas 57.3% were only FQs resistant and 15.9% were resistant to both FQs and SLIDs. Further DST of 225 isolates by liquid culture showed that 17% were sensitive to both FQs and SLIDs, 61.3% were only FQs resistant and 21.3% were resistant to both. The specificity for FQs and SLIDs was 92.31% and 100% whereas sensitivity was 100% respectively by GenoType MTBDRsl v.2.0 assay in direct sputum samples. Conclusions Our study clearly suggests that GenoType MTBDRsl v.2.0 assay is a reliable test for the rapid detection of resistance to second-line drugs after confirmation by GeneXpert MTB/RIF assay for RR-TB. Though, high rate FQ (ofloxacin) resistance was seen in our setting, moxifloxacin could be used as treatment option owing to very low resistance.

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