Hrev_master [page 64] [Healthcare in Low-resource Settings 2022; 10:10806] Evaluation of errors during susceptibility reporting of glycopeptide antibiotics for enterococcal isolates on sole basis of widely used disk diffusion test Vikas Saini, Nisha Goyal, Narendra Pal Singh, Meenakshi Goswami Department of Microbiology, University College of Medical Sciences & Guru Teg Bahadur Hospital, Delhi, India Abstract In resource limited settings, very often susceptibility reports glycopeptide antibi- otics are released only on the basis of disk diffusion test. Determination of MIC by either E-strips or microbroth dilution tests can be of paramount importance in check- ing the errors that may have incurred with the singular use of disk diffusion tests. Distribution of errors in Vancomycin and Teicoplanin susceptibility reporting by disk diffusion test was evaluated in comparison with MIC determination with reference method of E-strip. A total of 40 isolates of Enterococcus species were obtained during the study period. In vancomycin suscepti- bility reporting, very major errors, major errors and minor errors were observed in 33.3%, 5.4% and 5% instances respectively. For teicoplanin, major errors were observed in 5.3% of instances. E test can be used in conjunction with disk diffusion where resources are limited. We have observed that by using a combination of both disk dif- fusion and MIC determination by E-strip methods for glycopeptide antibiotics, a majority of reporting errors can be addressed. Introduction Enterococci were previously considered commensal organisms of little clinical importance but have emerged as serious nosocomial pathogens responsible for infections of bloodstream, meninges, uri- nary tract, biliary tract, wounds and endo- carditis.1 Commonly used glycopeptide antibiotics of vancomycin and teicoplanin have a crucial role in the management of severe infections due to enterococci in cases where patients are resistant or allergic to beta-lactam group of antibiotics.2-4 Plasmid- mediated resistance to these glycopeptide antibiotics, vancomycin and teicoplanin, was first detected in 1986.5,6 Resistant ente- rococcal strains responsible for coloniza- tion7-9 or infection10 have been isolated with increased incidence in current times. In vitro Vancomycin and teicoplanin susceptibility can be determined by disk diffusion, agar dilution, E-test, broth microdilution and automated antimicrobial susceptibility testing methods.11 The disk diffusion test is done by measuring the zones of growth inhibition that result when fixed concentrations of an antibiotic diffuse from impregnated disk onto an agar plate, which has previously been inoculated with the test organism. After proper incubation the inhibitory zones around the disk are measured and interpreted as resistant, inter- mediate and sensitive. The E-test combines diffusion with the ability to establish a Minimum Inhibitory Concentration (MIC) or breakpoint result. It is based on diffusion of a antimicrobial gra- dient from coated strips onto an agar surface inoculated with the test organism. MIC value is recorded directly from a scale on the strip in terms of µg/mL at the point where zone of growth inhibition intersects the strip.12 In resource limited settings, very often the susceptibility reports to these glycopep- tide antibiotics are released only on the basis of disk diffusion test. Determination of MIC by either E-strips or microbroth dilution tests can be of paramount impor- tance in checking the errors that may have incurred with the singular use of disk diffu- sion tests. The use of E-strips is less labor intensive and can be easily incorporated in routine testing even in high volume centers with restricted logistics. This study was planned to evaluate the errors during sus- ceptibility reporting of glycopeptide antibi- otics on sole basis of widely used disk dif- fusion test. Material and Methods This prospective study was conducted in the Department of Microbiology of a ter- tiary care hospital of Delhi over a period of six months extending from January 2022 to June 2022. All isolates of Enterococcus species obtained from wound aspirates were included in the study. All isolates of Enterococcus were iden- tified and tested for their antimicrobial sus- ceptibility patterns according to Clinical Laboratories Standards Institute (CLSI) guidelines. Antimicrobial susceptibility to Vancomycin & Teicoplanin in enterococcal isolates were performed using two methods, Kirby-Bauer disk diffusion technique and E-test method of Minimum Inhibitory Concentration (MIC) testing. CLSI has defined Susceptible (S), Intermediate (I), and Resistant (R) breakpoints for Vancomycin against enterococci for both disk diffusion (≥17 mm, S; 15-16 mm, I; <14 mm, R) dilution MIC testing (≤4µg/ml, S; 8-16 µg/ml, I; ≥32µg/ml, R) and for teicoplanin by disk diffusion (≥14 mm, S; 11-13 mm, I; ≤10 mm, R) and dilution MIC testing (≤8µg/ml, S; 16 µg/ml, I; ≥32µg/ml, R).13 E-test (Himedia Laboratories, Mumbai, India) was performed following CLSI guidelines.13 Healthcare in Low-resource Settings 2022; volume 10:10806 Correspondence: Nisha Goyal, Department of Microbiology, University College of Medical Sciences & Guru Teg Bahadur Hospital, Delhi. E-mail: drnishagoyalucms@gmail.com Key words: Enterococcus; glycopeptide antibiotics; vancomycin; teicoplanin. Funding: None. Conflict of interest: The Authors declare no conflict of interest. Availability of data and materials: All data generated or analyzed during this study are included in this published article. Ethics approval and consent to participate: Not applicable. Informed consent: Not applicable. This study used only the samples received in the lab for routine susceptibility testing and no other sample was collected for the purpose of this study. Patients were not identified or visited at any point of time. Received for publication: 20 August 2022. Revision received: 12 December 2022. Accepted for publication: 12 December 2022. This work is licensed under a Creative Commons Attribution 4.0 License (by-nc 4.0). ©Copyright: the Author(s), 2022 Licensee PAGEPress, Italy Healthcare in Low-resource Settings 2022; 10:10806 doi:10.4081/hls.2022.10806 Publisher's note: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affili- ated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guar- anteed or endorsed by the publisher. Non -co mmerc ial us e o nly [Healthcare in Low-resource Settings 2022; 10:10806] [page 65] Standard Kirby Bauer disk diffusion method was also used for determining the susceptibility of the isolates to the common- ly used antibiotics against Enterococcus spp. The antibiotic disks that were used to identify the susceptibility pattern of the Enterococcus spp. were ampicillin (10 µg), ciprofloxacin (5 µg), high level gentamicin (120 µg), tetracycline (30 µg), vancomycin (30 µg), teicoplanin (30 µg), linezolid (30 µg; Himedia Laboratories, Mumbai, India) and results were interpreted as per the latest CLSI guidelines.13 Distribution of errors in Vancomycin and Teicoplanin susceptibility reporting by disk diffusion test was evaluated in compar- ison with MIC determination with reference method of E-strip. Very major errors were defined as method in which the test result was susceptible and the reference method result was resistant. Major errors were defined as errors in which the test method result was resistant and the reference method result was susceptible, and minor errors were defined as those in which either method reported a result as intermediate and the other method reported the result as susceptible or resistant.14 Results A total of 40 isolates of Enterococcus species were obtained during the study peri- od. Table 1 shows the demographic profile of the patients with enterococcal isolates. Male is to female ratio was 0.67, showing a female preponderance. Over 50% of patients belonged to 20-40 years age-group. Out of 40 isolates, 38 isolates were Enterococcus faecalis and 2 isolates were E. faecium. Figure 1 depicts the distribution of susceptibility to commonly used antibi- otics among Enterococcus spp. isolates. 92.5% of Enterococcus spp. isolates were susceptible to Vancomycin and 95% were susceptible to teicoplanin by disk diffusion method. Tables 2 and 3 show the distribu- tion of errors in Vancomycin and teicoplanin susceptibility reporting by sin- gular use of disk diffusion method respec- tively. In vancomycin susceptibility report- ing, very major errors, major errors and minor errors were observed in 33.3%, 5.4% and 5% instances respectively (Table 2).For teicoplanin, major errors were observed in 5.3% instances (Table 3). Discussion Drug resistance has far reaching impacts on global healthcare. Various stud- ies have reported widely prevalent drug resistance among gram positive as well as gram negative organisms.15,16 In line with other studies, Enterococcus faecalis was predominant among enterococcal species in the present study.17,18 Though widely used, issue of false vancomycin resistance report- ing with disk diffusion method is a concern,19 prompting several manufacturers to issue alerts regarding the detection of vancomycin and teicoplanin resistant organisms. The current study reported very major, major and minor errors of 33.3%, 5.4% and 5% instances respectively in vancomycin susceptibility reporting by disk diffusion method. However, Tenover et al. observed Article Table 1. Demographic profile of patients with enterococcal isolates (n=40). Age group n (%) 0-10 years 2(5) 10-20 years 6(15) 20-30 years 13(32.50) 30-40 years 8(20) 40-50 years 3(7.5) 50-60 years 5(12.50) >60 years 3(7.5) Table 2. Distribution of errors in Vancomycin susceptibility reporting by singular use of disk diffusion method (n=40). Enterococci (n = 40) Method (no. reported) No. of very major errors/no. No. of major errors/no. No. of minor errors/total no. of resistant isolates (%) of susceptible isolates (%) of isolates (%) Disk diffusion 1/3 (33.3) 2/37 (5.4) 2/40 (5) Table 3. Distribution of errors in teicoplanin susceptibility reporting by singular use of disk diffusion method (n=40). Enterococci (n = 40) Method (no. reported) No. of very major errors/no. No. of major errors/no. No. of minor errors/total no. of resistant isolates (%) of susceptible isolates (%) of isolates (%) Disk diffusion 0 2/38 (5.26) 0 Figure 1. Distribution of susceptibility to commonly used antibiotics among Enterococcus spp. isolates. Non -co mmerc ial us e o nly [page 66] [Healthcare in Low-resource Settings 2022; 10:10806] no very major or major errors in their study while reporting and minor errors were lim- ited to ≤6%.20 The inception of intermediate and resistant breakpoints for vancomycin in enterococci (e.g., designating MIC of 8- 16µg/mL as intermediate and 32 µg/mL as resistant) would not address the high rates of very major errors as these breakpoints would still fail to capture several of the non- susceptible strains. Meanwhile, it would be difficult to lower the zone diameter for van- comycin to reclassify 17 mm (which cur- rently is in the susceptible range) as inter- mediate unless adequate data is available that demonstrates a lack of clinical efficacy. Thus, optimizing the reading of these tests particularly disk diffusion and E-test for enterococci is necessary to improve the accuracy of vancomycin and teicoplanin susceptibility results. E-test is superior to the disc diffusion method in detecting resis- tance among enterococcal isolates against glycopeptide antibiotics of vancomycin and teicoplanin.20 Though the gold standard test for detec- tion of resistance in Enterococcus spp. is broth dilution, its use in routine testing by manual antimicrobial susceptibility report- ing is difficult. E test can be used in con- junction to disc diffusion in where resources are limited. In present study we have observed that by using a combination of both disk diffusion and MIC determination by E-strip methods for glycopeptide antibi- otics, a majority of reporting errors can be inscribed. However, research involving wider population and large sample sizes is required to further back our findings. References 1. Murray BE. The life and times of the Enterococcus. Clin Microbiol Rev 1990;3:46-65. 2. Geraci JE, Wilson WR. Vancomycin therapy for infective endocarditis. Rev Infect Dis 1981;3:S250-8. 3. Gump DW. Vancomycin for treatment of bacterial meningitis. Rev Infect Dis 1981;3:S289-92. 4. Kirby WM. Vancomycin therapy in severe staphylococcal infections. Rev Infect Dis 1981;3:S236-9. 5. Leclercq R, Derlot E, Duval J, Courvalin P. Plasmid-mediated resis- tance to vancomycin and teicoplanin in Enterococcus faecium. New Engl J Med 1988;319:157-61. 6. Uttley AH, George RC, Naidoo J, et al. High-level vancomycin-resistant ente- rococci causing hospital infections. Epidemiol Infect 1989;103:173-81. 7. Williamson R, Al-Obeid S, Shlaes JH, et al. Inducible resistance to van- comycin in Enterococcus faecium D366. J Infect Dis 1989;159:1095-104. 8. Hirt H, Hall JW, Larson E, Gorr SU. A D-enantiomer of the antimicrobial pep- tide GL13K evades antimicrobial resis- tance in the Gram positive bacteria Enterococcus faecalis and Streptococcus gordonii. PLoS One 2018;13:e0194900. 9. Uttley AC. Vancomycin-resistant ente- rococci. Lancet 1988;2:57-8. 10. Uttley AH, George RC, Naidoo J, et al. 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Incidence and antimicrobial susceptibil- ity of enterococcal infections in tertiary care hospital. Intl J Microbiol Res 2018;10:1135–8. 16. Gupta V, Singla N, Behl P, et al. Antimicrobial susceptibility pattern of vancomycin resistant enterococci to newer antimicrobial agents. Indian J Med Res 2015;141:483. 17. Mathew SK. A profile of vancomycin- resistant enterococcal infections and a comparison of resistance detection methods. Indian J Microbiol Res 2018;5:408–14. 18. Rai S, Niranjan DK, Kaur T, et al. Detection of the classical G2576U mutation in linezolid resistant Staphylococcus aureus along with isola- tion of linezolid resistant Enterococcus faecium from a patient on short-term linezolid therapy: First report from India. Indian J Med Microbiol 2015;33:21–4. 19. Khalili H, Soltani R, Negahban S, et al. Reliability of disk diffusion test results for the antimicrobial susceptibility test- ing of nosocomial gram-positive microorganisms: is E-test method bet- ter? 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