|Year : 2021 | Volume
| Issue : 4 | Page : 379-387
Evaluation of drug susceptibility in nontuberculous mycobacteria using the SLOMYCO and RAPMYCO sensititre plates
Natalia Fernandes Garcia Carvalho, Carolina Salgado Pedace, Andreia Ramos Barbosa de Almeida, Fernanda Cristina dos Santos Simeão, Erica Chimara
Tuberculosis and Mycobacteriosis Laboratory, Institute Adolfo Lutz, São Paulo, Brazil
|Date of Submission||25-Sep-2021|
|Date of Decision||20-Oct-2021|
|Date of Acceptance||10-Nov-2021|
|Date of Web Publication||14-Dec-2021|
Tuberculosis Laboratory, Institute Adolfo Lutz, São Paulo
Source of Support: None, Conflict of Interest: None
Background: Non-tuberculous Mycobacteria (NTM) cause different forms of diseases. According to recent guideline by ATS/ERS/ESCMID/IDSA, drug susceptibility test (DST) is an important requirement to choose adequate treatment. The minimum inhibitory concentration (MIC) test is the recommended method. Sensititre SLOMYCO and RAPMYCO commercial panels were developed to perform mycobacteria DST easier. However, there are only two comparative studies between SLOMYCO and the MIC method and none for the RAPMYCO panel. The present study aimed to evaluate the Sensititre SLOMYCO and RAPMYCO plates in determining drug susceptibility compared to the gold standard method (MIC). Methods: The tests were carried out with clinical isolates received in the diagnostic routine of the Tuberculosis Laboratory at Institute Adolfo Lutz from the most frequent species in the state of São Paulo, Brazil. Reference strains were tested for repeatability and reproducibility analyses. MIC and Sensititre plates readings were compared with and without resazurin stain. Agreement between results was defined as MIC within the same dilution or dilution variation resulting the same category in both tests. Results were classified by categorical errors. Results: The RAPMYCO panel had 100% agreement for the drugs amikacin, doxycycline, ciprofloxacin and trimethoprim/sulfamethoxazole, 83.3% for clarithromycin and moxifloxacin and 60% for cefoxitin. The SLOMYCO panel had 80% agreement for amikacin and moxifloxacin and 60% for clarithromycin, rifabutin, rifampicin and ciprofloxacin. The repeatability and reproducibility with RAPMYCO and SLOMYCO plates showed a high level of agreement for the drugs tested, being higher with the use of resazurin. However, an evaluation on routine condition is needed. Conclusions: The present study found that the fewer steps in the tests with Sensititre plates and reading with resazurin allow its use with greater safety and efficiency in the laboratory routine. The results presented here will facilitate the execution of a validation for complete incorporation of Sensititre plates into a diagnostic routine.
Keywords: Drug susceptibility test, minimum inhibitory concentration, nontuberculous mycobacteria
|How to cite this article:|
Garcia Carvalho NF, Pedace CS, Barbosa de Almeida AR, dos Santos Simeão FC, Chimara E. Evaluation of drug susceptibility in nontuberculous mycobacteria using the SLOMYCO and RAPMYCO sensititre plates. Int J Mycobacteriol 2021;10:379-87
|How to cite this URL:|
Garcia Carvalho NF, Pedace CS, Barbosa de Almeida AR, dos Santos Simeão FC, Chimara E. Evaluation of drug susceptibility in nontuberculous mycobacteria using the SLOMYCO and RAPMYCO sensititre plates. Int J Mycobacteriol [serial online] 2021 [cited 2022 May 21];10:379-87. Available from: https://www.ijmyco.org/text.asp?2021/10/4/379/332365
| Introduction|| |
Nontuberculous mycobacteria (NTM) cause a wide variety of diseases, with pulmonary disease being the most frequent. Studies show there is a geographical variability in the distribution of NTM species and, since those species have the capacity to cause various diseases, such distribution has great clinical relevance., More than 190 species of NTM have already been described, among them, there are 25 species best known to cause mycobacteriosis. In the São Paulo State, the most populous state in Brazil, the species that frequently cause mycobacteriosis are Mycobacterium avium (MAC), M. kansasii, M. intracellulare, M. abscessus, M. chelonae, M. fortuitum, and M. peregrinum., NTM presents a significant difference in the drug susceptibility profile, mainly for therapeutic regimen recommended for tuberculosis. With the widespread increase of NTM incidence and the consequent increase in diseases caused by these mycobacteria, it is extremely important to perform the drug susceptibility test (DST) in a simple and quick way to evaluate drugs that can be used in the treatment. The correct choice is essential since the treatments last for at least 12 months and the use of a drug in which the agent is resistant can cost time and the patient's quality of life.,
The minimum inhibitory concentration test (MIC) is the method recommended by the National Committee for Clinical Laboratory Standards Institute. MIC is based on the lowest concentration of an antimicrobial agent that prevents the visible growth of a microorganism in DST by dilution in broth. For NTM, standardization is only available for species that have clinical significance.,, The MIC, being an in house technique, requires a lot of attention to avoid errors in drug concentrations and in all stages of execution. The performance of this methodology is recommended only for laboratories with extensive experience in the NTM diagnosis., To obtain greater precision in the results, resazurin can be used in microplates as a colorimetric test.,,
To provide faster results and to reduce possible flaws in the pipetting and storage of drugs, commercial plates were developed. The Trek Sensititre SLOMYCO and RAPMYCO (Trek Diagnostics/Thermo Fisher, Bremen, Germany) plates are microdilution assays containing lyophilized drugs and configured for the MIC determination of slow-growing (SLOMYCO) and fast-growing (RAPMYCO) mycobacteria species. The reading of plates can be automated using Sensititre® Vizion® equipment (Trek Diagnostic Systems), which allows digital capture of the image and is specific for reading the Sensititre plates. However, only two comparative studies have been carried out to compare SLOMYCO and the standard MIC method., Babady et al. compared the results obtained in the SLOMYCO with MIC, testing only two species, MAC and M. intracellulare and using only clarithromycin. The agreement between methods was 93%, with a kappa value of 0.87, showing good agreement. Chazel et al. also compared the SLOMYCO panel with the MIC method. However, the study was performed only for species M. marinum. In this study, the agreement was 100% for amikacin, ciprofloxacin, clarithromycin, moxifloxacin, rifabutin, sulfamethoxazole-trimethropim, 98% for ethambutol, and 96% for rifampicin, showing that it is a diagnostic alternative for M. marinum, except for doxycycline.
For RAPMYCO panel, no comparative studies were carried out using the mycobacterial species commonly causing mycobacterioses. Cavusoglu et al. evaluated the susceptibility profile of M. abscessus, M. chelonae, M. fortuitum, and M. peregrinum using the RAPMYCO panel but did not compare the results with any other method.
Against this scenario, the present study aimed to evaluate the Sensititre SLOMYCO and RAPMYCO plates in determining drug susceptibility in clinical NTM isolates in a routine laboratory, using MIC as the gold standard.
| Methods|| |
All stages of this study were carried out at the Biosafety Level 3 Laboratory (BSL3) at the Tuberculosis and Mycobacteriosis Laboratory of the Institute Adolfo Lutz (IAL).
This study was conducted in three stages in order to standardize the method Sensititre, to compare with the standard method (in house MIC), and to assess reproducibility and repeatability. To achieve goals, an algorithm was designed for each step, shown in [Figure 1].
Reference strains MAC ATCC 25291 and M. abscessus ATCC 19977 were used to perform Stage 1 and 3 and M. kansasii ATCC 12478 for Stage 3.
The tests were conducted with clinical isolates, received in the IAL diagnostic routine, of the seven most frequent species isolated in Sao Paulo State, namely, MAC, M. intracellulare, M. kansasii, M. abscessus subsp. abscessus, M. abscessus subsp. bolletii, M. chelonae, M. peregrinum, and M. fortuitum. The isolates, chosen at random, were identified by the PRA-hsp65 technique and biochemical tests.
Minimum inhibitory concentration in Mueller-Hinton cation adjusted broth
To obtain the reading with the gold standard method (in house MIC), the test was performed with the standard protocol described by CLSI. The isolates were tested in two different plate designs, one for slow-growing mycobacteria (SGM) and one for rapid-growing mycobacteria (RGM) [[Figure 2] and [Figure 3], respectively]. Drugs were diluted according to document M100-S22, aliquoted, and stored at −20°C. 96-well microplates were prepared using Mueller–Hinton cation-adjusted broth (MHC) and the drugs used in the treatment of mycobacteriosis were added. The bacterial inoculum was prepared in MHC, at the optical density of the 0.5 McFarland scale, and added to each well of the plate, reaching the final concentration of 1.5 × 105 UFC/ml. Visual readings were taken after 3 days of incubation for RGM and 7 days for SGM. After reading, resazurin was added to each well, as described by Carvalho et al., MIC values obtained were interpreted according to Document M62.
|Figure 2: Plate design for the minimum inhibitory concentration test for SGM. A-H: Decreasing concentrations (μg/mL), in proportion 2, of the drugs distributed in columns 2-10; CC: Growth control; CM: Media sterility control; INH: Isoniazid; RF: Rifampicin; EMB: Ethambutol; STR: Streptomycin; RB: Rifabutin; CIP: Ciprofloxacin; AK: Amikacin; CLA: Clarithromycin; MFX: Moxifloxacin|
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|Figure 3: Plate design for the minimum inhibitory concentration test for RGM. A-H: Range used for each drug concentration (μg/mL); CC: Growth control; CM: Media sterility control; AMK: Amikacin; FOX: Cefoxitin; CIP: Ciprofloxacin; CLA: Clarithromycin; DOX: Doxycycline; MFX: Moxifloxacin; SXT: Trimethoprim/sulfamethoxazole|
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Minimum inhibitory concentration in Sensititre SLOMYCO and RAPMYCO plates
The commercial plates SLOMYCO and RAPMYCO [Figure 4] and [Figure 5] were prepared according to the manufacturer's instructions. Readings were taken after 3 days for RGM and 7 days for SGM, using the Sensititre® Vizion® equipment, by bright indirect lighting against a dark background. After this reading, the resazurin dye was added, and the plates were incubated for another 24 h. A new reading was held on the same equipment using indirect lighting against a white background. The MIC values obtained were interpreted according to Document M62.
For repeatability analysis, intraday precision was evaluated, in which ten repetitions were performed in a single day (ten plates of the same sample numbered from 1 to 10).
For reproducibility, the intermediate precision was evaluated, in which nine repetitions were performed in 9 days, with one plate for each strain per day.
The methodology used to prepare plates and inoculation was the same described for the tests with clinical isolates and readings were performed with and without resazurin for comparison.
The MIC and Sensititre plates readings were compared. Analyses were made only for drugs that were available in the IAL susceptibility test. The agreement between the results was defined as MIC within the same dilution or variation of a dilution within the same category in both tests.
Categorical errors were classified into three types: Minor (intermediate resistance versus a susceptible or resistant result), major (MIC, susceptible result; sensititre, resistant result [false resistant]), or very major (MIC, resistant result; sensititre, susceptible result [susceptible false]).
| Results|| |
Comparison between minimum inhibitory concentration and Sensititre methods using reference strains
The tests with sensititre plates and microplates prepared in house were tested and compared initially with reference strains to verify viability [Table 1].
|Table 1: Minimum inhibitory concentration of reference strains, obtained with the use of sensititre plates, and microplates in house after reading with resazurin|
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Both methods showed concordant results when the reference strain M. abscessus ATCC 19977 was tested, except for moxifloxacin. This disagreement was categorized as a minor error, since, in addition to the categorical difference, the results have a difference of two logs.
For the MAC ATCC 25291 strain, the methods were concordant for all drugs recommended by CLSI, amikacin, clarithromycin, and moxifloxacin. The MIC values obtained for MAC by both methods were concordant for isoniazid, ciprofloxacin, ethambutol, and rifabutin and discordant for streptomycin and rifampicin, whose MIC in the in house technique was four times higher than in the SLOMYCO plate for both drugs.
Regarding the reference strain M. kansasii ATCC 12478, the results were concordant for both methods for amikacin, moxifloxacin, and rifabutin. Discordant results for ciprofloxacin were considered to be a very serious error, due to the resistance presented in the in house technique and susceptibility on the SLOMYCO plate (false susceptibility). Clarithromycin and rifampicin showed discordant results, presenting susceptibility in the gold standard technique and resistance in the commercial test, considered, therefore, a false resistance, a serious error for both drugs.
Ethambutol, isoniazid, and streptomycin do not have established values for determining susceptibility to M. kansasii. However, the results obtained by both methods were discordant for the three drugs, whose MIC in the gold standard technique was two, three, and four times lower than in SLOMYCO for ethambutol, isoniazid, and streptomycin, respectively.
Comparison between in house and sensititre plates using clinical isolates
For clinical isolates of M. a. abscessus, M. a. bolletii, and M. peregrinum results were concordant for all drugs between the two methods [Table 2]. For M. chelonae, only cefoxitin presented resistance in the gold standard method and intermediate resistance by RAPMYCO, with a difference of three logs, classified as a minor error. For M. fortuitum, cefoxitin, ciprofloxacin, and clarithromycin results disagreed between methods, but only the results of cefoxitin and clarithromycin were classified as minor errors, with a difference of two dilution logs for both drugs.
|Table 2: Minimum inhibitory concentration of rapid-growing clinical isolates obtained with the use in house and sensititre plate for testing fast growing mycobacteria plates after reading with resazurin|
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For clinical isolates of M. intracelullare, the results were concordant only for moxifloxacin [Table 3]. For amikacin and clarithromycin, the errors were considered very major, due to false susceptibility result in the SLOMYCO plate and high resistance by the in house plate. Ethambutol, isoniazid, rifampicin, and streptomycin, despite not have established values for M. intracellulare, showed agreement between the two methods. In contrast, ciprofloxacin and rifabutin showed results four and five times higher, respectively, by the commercial method compared to in house.
|Table 3: Minimum inhibitory concentration of slow-growing clinical isolates obtained with the use of in house and sensititre plate for testing slow-growing mycobacteria plates after reading with resazurin|
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Regarding M. kansasii, MIC values and susceptibility criteria were consistent for all drugs by both methods, except for moxifloxacin, with a susceptible result in the in house method and intermediate on the SLOMYCO panel, a difference of three dilution logs, classified as minor error.
Among the drugs that do not have established susceptibility values for M. kansasii, results were concordant, and isoniazid and streptomycin were discordant, four times lower for SLOMYCO and three times higher for SLOMYCO, respectively.
The results were concordant for amikacin and clarithromycin and discordant for moxifloxacin in the tests with MAC, with disagreement categorized as a very major error. Among the drugs with no established cutoff points, streptomycin was the only one that presented concordant results in both methods, while ciprofloxacin and isoniazid drugs showed a result five times higher by in house plates compared to the SLOMYCO plates. For rifampicin, the gold standard method showed three times greater results compared to the commercial panel, and for the drug rifabutin, the results were twice less in the in house method compared to SLOMYCO.
Evaluation of resazurin readings
The concordance between RAPMYCO plates with visual reading and reading after addition of resazurin was below 100% only for trimethoprim/sulfamethoxazole and clarithromycin [Table 4]. These drugs showed only one isolate with discordant results between the tested RGM isolates and the reference strain M. abscessus ATCC 19977.
|Table 4: Agreement between results from Sensititre plate for testing fast-growing mycobacteria panel, with and without resazurin, and between the Sensititre plate for testing fast-growing mycobacteria methods and in house broth microdilution after visual and resazurin readings, using rapid-growing mycobacteria isolates|
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One isolate showed susceptibility for clarithromycin in visual reading and intermediate resistance after resazurin addition, which received the lowest error rating. Only trimethoprim/sulfamethoxazole had a result categorized as a very major error, due to an isolate that showed false susceptibility in visual reading and resistance after addition of resazurin.
In the comparison between the results obtained after readings with resazurin, 100% agreement was observed in the readings for amikacin, doxycycline, ciprofloxacin, and trimethoprim/sulfamethoxazole [Table 4]. For clarithromycin, moxifloxacin, and cefoxitin, discrepancies in results were classified as minor errors.
When comparing results obtained after reading SLOMYCO plates, only amikacin had 100% agreement between the five samples of SGM studied [Table 5]. The agreement was 80% for amikacin and moxifloxacin and 60% for clarithromycin, rifabutin, rifampicin, and ciprofloxacin [Table 5] for all SGM isolates. Comparing visual and resazurin reading on SLOMYCO and in house plates, rifampicin, ciprofloxacin, and ethambutol showed a higher agreement, without changing for other drugs.
|Table 5: Agreement between results from sensititre plate for testing slow-growing mycobacteria panel, with and without resazurin, and between the sensititre plate for testing slow-growing mycobacteria methods and in house broth microdilution after visual and resazurin readings, using slow-growing mycobacteria isolates|
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Precision study results
Repeatability and reproducibility were analyzed for RAPMYCO and SLOMYCO plates with visual readings and with the use of resazurin. In the repeatability of the RAPMYCO panels with visual readings, it was observed that the agreement was above 90%, with the exception for clarithromycin and trimethoprim/sulfamethoxazole, which presented an agreement of 60%. However, the repeatability of RAPMYCO with resazurin was 100% consistent for all drugs, except for clarithromycin which increased to 80%.
The reproducibility of RAPMYCO panels with visual readings and resazurin showed an agreement above 90%, except for imipenem, with a 50% agreement on visual reading, and an increase to 90% in reading with resazurin [Table 6].
|Table 6: Percentage of agreement for repeatability and reproducibility for Sensititre plate for testing fast-growing mycobacteria in readings with and without resazurin|
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The repeatability of SLOMYCO panels with visual readings and resazurin showed high agreement for all drugs, except for clarithromycin and linezolid, which presented 80% and 70% for both readings, respectively. The reproducibility of SLOMYCO panels with visual reading showed high levels of agreement for drugs in general, 80% for clarithromycin, 70% for trimethoprim/sulfamethoxazole, and 60% for linezolid and rifabutin. Reproducibility with resazurin showed high agreement for all drugs, except for clarithromycin and rifabutin, which had 80% agreement [Table 7].
|Table 7: Percentage of agreement for repeatability and reproducibility for Sensititre plate for testing slow-growing mycobacteria in readings with and without resazurin|
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| Discussion|| |
DST for NTM is an important requirement since there is no established standard treatment for these microorganisms. A recent guideline, published by American and European Thoracic Societies, reinforces the needed to know the susceptibility profile for the establishment of the adequate therapy. The methodology used for DST is the MIC by microdilution in broth, recommended by CLSI, which requires many steps to be performed in house and a professional trained in reading and interpretation.
The Tuberculosis and Mycobacteriosis Laboratory of the Adolfo Lutz Institute performs DST for NTM using microdilution in broth with resazurin as a routine for clinical isolates collected from patients in the State of São Paulo, Brazil, since 2015.
The present work evaluated the use of a commercial methodology for susceptibility testing and the differential of this evaluation was the use of resazurin dye in Sensititre plates, which allows laboratories that do not have the equipment for reading to perform it in a simple way. Data obtained in the present study showed resazurin does not alter the readings and provide more reliable results. In addition, it was observed that agreement between in house and Sensititre plates increases with the use of resazurin.
Comparing the MIC values and the categorical criteria between the in house and Sensititre plates for the reference strains, an agreement of 85.7% was observed for M. abscessus ATCC 19977, with only one discrepancy considered as a minor error for moxifloxacin, while for the SGM reference strains, some conflicting results were observed. For the reference strain MAC ATCC 25291, an agreement of 77.7% was observed, and for the reference strain M. kansasii ATCC 12478, only three concordant results among nine tested drugs were observed (33.3%).
When the clinical isolates were tested, a high agreement was observed between the in house method and RAPMYCO panel, with conflicting results considered as minor errors. These type of errors occurred at dilutions above or below the cutoff, which according to Trollip et al. are characterized as borderline MIC.
On the other hand, the agreement between in house method and SLOMYCO panel showed many discrepancies considered as major errors and very major errors. This can be attributed to the fact that SGM is more demanding, requiring longer incubation time and the use of supplementation (OADC). The disagreements found for the SLOMYCO panel may also be associated with the fact that most drugs present in this commercial plate do not have susceptibility values established by CLSI for the MAC complex, a complex that makes up 60% of the SGM isolates in this study. Many of the results observed when comparing the methods could be within the same category, even with more than one log of difference. However, due to the lack of established categories, the difference of more than one concentration well causes the results to be reported as discordant.
CLSI states on its MAC breakpoints page that, although the drugs ethambutol, rifampicin, and rifabutin are clinically useful, breakpoints for determining susceptibility or resistance for these agents have not been established because previous studies have shown that there has been no correlation between in vitro MIC results and clinical response in patients with MAC., However, in our routine, these drugs are tested for purposes of epidemiological analysis. The drug streptomycin, in turn, can be used in place of amikacin; however, no study has correlated the MIC values with the clinical response in patients affected by MAC, for this reason, if the drug streptomycin is tested, only the value of the MIC should be reported.
In addition to these issues that can be attributed to disagreements in the SLOMYCO panel, another important point that must be taken into account is that when preparing microplates internally, great care is needed to avoid pipetting errors, which can result in changes in drug concentrations. According to the quality standards, the final result of the susceptibility tests is significantly influenced by the methodology, which must be carefully controlled to obtain reproducible results (intra and interlaboratory).
Nikolayevskyy et al. conducted a study to verify the methodologies used for DST to NTM on 31 laboratories and observed a significant heterogeneity among results. The authors explained that this difference was not due to the use of different methods, but because of variants such as inoculum preparation, incubation time, and subjective interpretation of bacterial growth.
Although we use the method recommended by CLSI, given the difficulties in which each step of the test can involve an error adding a sequence of bias, these errors can be drastically reduced with the use of ready-made commercial plates, as they have lyophilized drugs. In addition, an interesting control measure to apply in these tests would be the use of equipment that inoculates the drugs and also reads them, generating more reliable results and interpretations.
The limitation of this study is that it does not have a cost analysis. However, considering the greater chance of error in the in house test and the need to repeat the test, it is likely that the commercial plate is cost effective since provide more correct results testing the isolate once.
The repeatability and reproducibility for RAPMYCO and SLOMYCO showed a high level of agreement, mainly with the use of resazurin. According to these observations, RAPMYCO and SLOMYCO presented results with good repeatability and reproducibility, suggesting their implementation in a DST routine for NTM. However, more comparative studies are needed, mainly for the SLOMYCO panel, to assess the disagreements obtained.
| Conclusion|| |
The present work evaluated the use of Sensititre plates and found that the smaller number of steps for its execution and reading with resazurin allow its use with greater safety and efficiency on the DST routine for NTM. Although the study has a limited number of isolates tested, the results presented here will facilitate the execution of a validation, which should be performed for the complete incorporation of commercial plates in a diagnostic routine.
The present study was approved by the Scientific and Research Ethics Committees of Institute Adolfo Lutz under number 30-I/2016.
Financial support and sponsorship
This work was supported by Thermo Fisher Scientific who supplied the Sensititre medium and plates. The authors have directed and are fully responsible for all content and editorial decisions.
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6], [Table 7]