|Year : 2018 | Volume
| Issue : 2 | Page : 93-97
RAPMYCO: Mitigating conventional broth microdilution woes
Gurpreet Singh Bhalla1, Naveen Grover2, Lavan Singh1, Manbeer S Sarao3, Dinesh Kalra4, Chetna Pandey2
1 Department of Laboratory Sciences, Army Hospital (R & R), New Delhi, India
2 Department of Microbiology, AFMC, Pune, Maharashtra, India
3 Division of Infectious Diseases, Detroit Medical Centre, Detroit, MI, USA
4 Department of Microbiology, Command Hospital (WC), Chandimandir, Panchkula, Haryana, India
|Date of Submission||15-Dec-2017|
|Date of Acceptance||26-Apr-2018|
|Date of Web Publication||13-Aug-2018|
Dr. Naveen Grover
Department of Microbiology, AFMC, Pune, Maharashtra
Source of Support: None, Conflict of Interest: None
Aim: Nontuberculous mycobacteria (NTM) are proven pathogens causing a plethora of diseases in humans. Various methods are available for their identification and susceptibility testing. Since their susceptibility varies with species, it becomes imperative to perform drug susceptibility testing. Various methods are available, of which broth microdilution is recommended by the Clinical and Laboratory Standards Institute (CLSI). We report our results after using RAPMYCO, commercially available, predosed, ready-to-use broth-microdilution plate. Materials and Methods: A total of 33 isolates of NTM were tested using the RAPMYCO panel for susceptibility against amikacin, cefoxitin, ciprofloxacin, clarithromycin, doxycycline, imipenem, linezolid, trimethoprim + sulfamethoxazole, tobramycin, and tigecycline, and the results were interpreted as per the CLSI guidelines. Results and Conclusion: Minimum inhibitory concentration results of conventional broth microdilution correlated well with those of RAPMYCO. All Mycobacterium fortuitum and Mycobacterium chelonae isolates were susceptible to amikacin and tobramycin.Good susceptibility was observed towards clarithromycin for all isolates; some degree of susceptibility was observed for quinolones and linezolid. High degree of resistance was seen for cefoxitin, doxycycline, and trimethoprim + sulfamethoxazole. Mycobacterium abscessus was the most resistant. RAPMYCO was simple, easy, and saved precious person-hours as compared to conventional broth microdilution.
Keywords: Broth microdilution, nontuberculous mycobacteria, RAPMYCO
|How to cite this article:|
Bhalla GS, Grover N, Singh L, Sarao MS, Kalra D, Pandey C. RAPMYCO: Mitigating conventional broth microdilution woes. J Health Res Rev 2018;5:93-7
|How to cite this URL:|
Bhalla GS, Grover N, Singh L, Sarao MS, Kalra D, Pandey C. RAPMYCO: Mitigating conventional broth microdilution woes. J Health Res Rev [serial online] 2018 [cited 2019 May 19];5:93-7. Available from: http://www.jhrr.org/text.asp?2018/5/2/93/238860
| Introduction|| |
Nontuberculous mycobacteria (NTM) are composed of mycobacterial species other than the Mycobacterium tuberculosis complex, are widely distributed in nature, and were initially thought to be mere contaminants when isolated. However, in the last few years, they have been reported to cause a myriad of diseases ranging from hospital-acquired infections to diseases following environmental exposures such as hurricanes and tsunamis,, both in immunocompetent and immunocompromised individuals. Epidemiological data from the Infectious Diseases Society of American Emerging Infections Network and information from referral centers suggest that NTM infections have been consistently on the rise. Almost all diseases caused by rapidly growing mycobacteria (RGM), including surgical wound infections,,, skin and soft tissue infections,, implant-associated infections,, and catheter-associated infections  in humans are due to Mycobacterium fortuitum, Mycobacterium chelonae, and Mycobacterium abscessus.
Different species of NTM have different susceptibility to antimicrobial agents; even individual strains are reported to have varying susceptibility. It is reiterated that NTM are not susceptible to routinely used antitubercular drugs. Hence, it becomes imperative to perform drug susceptibility testing on the isolates before starting therapy to avoid irrational use.
Various methods of drug susceptibility testing include agar-based methods, broth microdilution, alamarBlue assay, E-test, flow cytometry, radiometric methods, disc elution methods, and diffusion methods.,, Broth microdilution is the method recommended by the Clinical and Laboratory Standards Institute (CLSI). We present here our experience with the use of commercially available RAPMYCO broth microdilution minimum inhibitory concentration (MIC) panel (SENSITITRE ®, Trek Diagnostic Systems, UK) for testing of 33 isolates of RGM isolated from surgical site infections (SSIs) at a tertiary care center.
| Materials and Methods|| |
The study was prospective with a duration of 2 years. All patients (n = 303) fulfilling the criteria of SSI (as per the CDC guidelines) of the total 7675 surgeries performed during the study period were included in the study for the isolation of NTM. Informed consent from patients was obtained before collecting the samples followed by clearance from the Institutional Ethical Committee (IEC/03Dec2013 AFMC dated December 10, 2013). No clinical trials or new drug testing was done in this study. Relevant samples were collected aseptically and examined by Gram's stain and Ziehl–Neelsen stain. Samples were simultaneously inoculated on Lowenstein–Jensen (LJ) media along with routinely used 5% sheep blood agar and MacConkey media. Isolates were identified as NTM by the standard phenotypic detection methods.
Following the isolation, antibiotic susceptibility profile was performed as per the CLSI guidelines  by broth microdilution method using RAPMYCO. This method is validated for the susceptibility testing of RGM including M. fortuitum group (M. fortuitum, Mycobacterium peregrinum, M. fortuitum third biovariant complex), M. chelonae, M. abscessus, Mycobacterium mucogenicum, and Mycobacterium smegmatis group (M. smegmatis, Mycobacterium goodii, Mycobacterium wolinskyi), Nocardia spp., and other aerobic actinomycetes.
Each plate is dosed with antimicrobial agents at appropriate dilutions [Figure 1]. Results can be read manually as turbidity/button formation [Figure 2].
Confluent portion of active growth from LJ media was swept and emulsified in sterile water and adjusted to a 0.5 McFarland standard (without lump formation). From this suspension, 50 μl was transferred into a tube of cation-adjusted Mueller-Hinton broth with TES buffer to give an inoculum of 5 × 105 CFU/mL, and 100 μL of this inoculum was transferred to each well within 30 min. Growth was checked after incubation at 30°C in a non-CO2 incubator for 72 h, and if poor, re-incubation was done for up to a further 48 h (4–5 days may be required for isolates of M. chelonae and M. abscessus).
To interpret, growth control wells were read first (if they show no growth, results are invalid). The MIC was noted as the lowest concentration that completely inhibited the growth, except for sulfonamides, whereas the MIC was read as the lowest concentration that inhibited 80% growth compared to the positive control. Purity was checked by subculturing growth declaring an invalid test result if mixed culture was detected.
The susceptibility testing in the present study was done for amikacin, cefoxitin, ciprofloxacin, clarithromycin, doxycycline, imipenem, linezolid, trimethoprim + sulfamethoxazole, tobramycin, and tigecycline. MIC values obtained were interpreted as per the CLSI breakpoints [Table 1].
|Table 1: Clinical and Laboratory Standards Institute recommended breakpoints of various drugs for nontuberculous mycobacteria|
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| Results|| |
A total of 33 NTM were isolated. All isolates were RGM with most common isolate being M. fortuitum (n = 17) followed by M. abscessus (n = 13) and M. chelonae (n = 3). [Table 2], [Table 3], [Table 4] show the MIC of M. fortuitum, M. abscessus, and M. chelonae isolates, respectively, to various drugs. The summary of susceptibility pattern of all the NTM isolates is given in [Table 5]. The same MIC values were obtained both by conventional broth microdilution and by RAPMYCO.
|Table 2: Interpretation and minimum inhibitory concentration of Mycobacterium fortuitum isolates (n=17) in μg/mL|
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|Table 3: Interpretation and minimum inhibitory concentration of Mycobacterium abscessus isolates (n=13) in μg/mL|
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|Table 4: Interpretation and minimum inhibitory concentration of Mycobacterium chelonae isolates (n=3) in μg/mL|
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|Table 5: Summary of susceptibility pattern of all the isolates of nontuberculous mycobacteria|
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| Discussion|| |
Four decades ago, no specific guidelines for drug susceptibility testing of NTM existed. Since then, more workers began testing standard methods recommended for testing of RGM (Enterobacteriaceae, etc.) using agar and broth dilution techniques. Broth was later advised because M. chelonae grew better in broth than on agar, especially if no supplements such as oleic acid-albumin-dextrose-catalase were added.
In the present study, M. fortuitum (51.51%) was the most common isolate as reported by other workers.,
The susceptibility of NTM isolated in the present study shows that all M. fortuitum isolates were susceptible to amikacin with MIC of ≤16 μg/mL. 94.1% of isolates were susceptible to ciprofloxacin, clarithromycin, and linezolid. Only 43% of isolates were sensitive to imipenem. Fifty-three percent were resistant to doxycycline and trimethoprim-sulfamethoxazole.
As for M. abscessus, 92.3% isolates were susceptible to amikacin with 84.6% susceptibility to clarithromycin, 77% to ciprofloxacin, and only 53.8% to linezolid. 92.3% isolates were resistant to doxycycline and trimethoprim-sulfamethoxazole and 84.6% to cefoxitin.
Finally, all M. chelonae isolates were susceptible to amikacin, clarithromycin, linezolid, and tobramycin. All isolates were resistant to trimethoprim-sulfamethoxazole and 66.7% to cefoxitin, ciprofloxacin, and doxycycline.
Findings of the present study correlate with the previous studies as follows:,,,,,
- M. fortuitum demonstrating least resistance to antimicrobial agents
- Amikacin (tobramycin for M. chelonae) and clarithromycin being the choice of empirical therapy of NTM infections
- High degree of resistance by all isolates to trimethoprim-sulfamethoxazole, doxycycline, and cefoxitin
- Even though breakpoints have not been mentioned, tigecycline emerges as a potential therapeutic agent as evidenced by its low MIC values.
The current literature advises against the use of monotherapies for the treatment of infections caused by NTM to avoid the emergence of resistance. Workers have also tested and reported the synergistic effect of clofazimine with amikacin and clarithromycin; however, this combination is not yet mentioned in the CLSI guidelines or available in RAPMYCO and was not tested.,
Broth microdilution is the method for susceptibility testing of NTM recommended by CLSI, but it is a time-consuming process requiring utmost precision and dedication for the preparation of stock and work solutions ensuring sterility throughout the process. Proper storage is another important factor which affects the reliability of results. Finally, performing tests on multiple isolates can be very tiring, thereby decreasing the efficiency of the worker resulting in skip or wrong wells.
| Conclusion|| |
RAPMYCO can be used straight from the box and saves numerous person-hours used in the preparation of solutions, and its use does not require much technical training. The results are reliable and reproducible, and it includes the drugs recommended by the CLSI guidelines. Thus, RAPMYCO is an effective way to perform broth microdilution for susceptibility testing of NTM isolates in a routine microbiology laboratory.
More studies should be performed with greater number of NTM isolates as the limitation of the present study was less number of isolates. Future studies along with speciation and isolate typing will also help understand the species-based antibiotic susceptibility patterns of clinical isolates of NTM, thereby helping to formulate therapeutic guidelines for NTM infections and mechanism of drug resistance.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]