Abstract
Purpose.
Neisseria gonorrhoeae is a sexually transmitted bacterial pathogen that continues to evolve to become resistant to known antibiotics. In preparing for potential emergence, the Centers for Disease Control and Prevention recommends that clinical laboratories maintain or develop protocols to assess antibiotic susceptibly for this organism. This study examines the intra-laboratory variability of using the Etest method to provide consistent MIC values for N. gonorrhoeae and also compared the results of the Etest to known agar dilution MIC values.
Methodology.
Clinical N. gonorrhoeae isolates, 100 paired duplicates, were tested by eight laboratories for antibiotic susceptibility to ceftriaxone, cefixime and azithromycin using Etest strips.
Results/Key findings.
Overall, >80 % of the paired Etest MIC values were within one log2 dilution of the replicate. When compared to the agar dilution reference method, the cefixime Etest MIC values were consistently underreported by one dilution (seven laboratories) or two dilutions (one laboratory). The azithromycin Etest MIC values agreed 90.7 % with the agar dilution MIC values while the agreement with ceftriaxone was 90.9 %.
Conclusion.
Overall, the Etest method yielded reproducible MIC values within each laboratory with the azithromycin and ceftriaxone MIC results consistent to the reference agar dilution method while the cefixime result tended to provide a lower MIC value.
Keywords: Gonorrhea, antimicrobial susceptibility, Etest accuracy, Etest reproducibility
INTRODUCTION
Neisseria gonorrhoeae is a sexually transmitted bacterial pathogen that is responsible for the second most common notifiable communicable disease in the United States with 334 826 cases reported to the Centers for Disease Control and Prevention (CDC) in 2012 [1]. Women are typically asymptomatic until complications such as salpingitis develop [2]. In men, the majority of urethral infections cause urethritis with painful urination and, less commonly, epididymitis [2]. In rare cases, severe complications such as septic arthritis, endocarditis and meningitis result from disseminated gonococcal infections. Antibiotics used to treat N. gonorrhoeae become less effective as the organism develops or acquires resistance through genetic evolution that alter phenotypic characteristics [3]. Current CDC treatment recommendations for uncomplicated N. gonorrhoeae infections of the cervix, urethra, rectum and oropharynx is combination therapy with ceftriaxone 250 mg as a single intramuscular dose, plus either azithromycin 1 g orally in a single dose or doxycycline 100 mg orally twice daily for 7 days [4]. If ceftriaxone is not available, 400 mg cefixime orally in a single dose may be used as an alternative in combination with azithromycin or doxycycline for cervical, urethral or rectal infections. Antibiotic susceptibility data from the Gonococcal Isolate Surveillance Project (GISP) indicate that N. gonorrhoeae isolates in the United States remain susceptible to current recommendations [5]. However, treatment failures and decreased susceptibly to these antibiotics have been reported in Austria, France, Japan, Norway, Spain and the United Kingdom [6–10].
Microbiologic assessment using viable isolates and culture-based susceptibility testing are essential to investigate cases of suspect treatment failures because assays that detect molecular resistance deteminants to antibiotics recommended for treatment are still being developed and/or evaluated [11, 12]. Laboratory capacity for N. gonorrhoeae culture and antibiotic susceptibility testing is critical to monitor for emerging resistance yet nucleic acid amplification tests have largely replaced culture in most clinical laboratories [13]. Susceptibility of N. gonorrhoeae to antimicrobial agents can be determined by the disk diffusion, Etest (bioMérieux, Durham, NC, USA), or agar dilution methods. The Clinical Laboratory Standards Institute (CLSI) has established interpretative inhibition zone diameters to permit classification of an isolate as being susceptible, intermediate or resistant to penicillin, parental and oral cephems, tetracyclines, fluoroquinolones and spectinomycin [14]. However, the utility of disk diffusion is low since there are no interpretative criteria for N. gonorrhoeae susceptibility to azithromycin by this method. Agar dilution is the gold standard method for antimicrobial susceptibility testing to provide a MIC value of antimicrobial agents tested against N. gonorrhoeae but might be too difficult to perform in laboratories with limited capacity and low testing volumes [15]. Etest strips can also provide a quantitative MIC value and have been assessed in a few studies with N. gonorrhoeae and are simpler tests to perform [16–23]. Still there is little data on the implementation of Etest strips in clinical laboratories tasked with addressing antibiotic susceptibility testing for N. gonorrhoeae. We investigated the accuracy and reproducibility of Etest strips to assess the antibiotic susceptibility of N. gonorrhoeae by comparing intra-laboratory variability and MIC values to those from agar dilution.
METHODS
N. gonorrhoeae isolates and culture conditions
A total of 100 clinical N. gonorrhoeae isolates, obtained from submissions to the CDC for reference antibiotic susceptibility testing between 2009 and 2012, were included in this evaluation. These isolates, which were tested in duplicate at an interval of 3 weeks or greater using the agar dilution method had reproducible MIC values for azithromycin, cefixime and ceftriaxone. The bacterial identification and MIC values were confirmed by the CDC prior to being incorporated into the assessment panel. Each isolate was subsequently duplicated, uniquely coded and frozen at −70 °C in 1.0 ml cryovials containing trypticase soy broth with 20 % glycerol. The isolates were maintained in a frozen state using dry ice while being shipped to eight United States Public Health Laboratories. The participating laboratories were unaware of the previous antibiotic susceptibility results and that the 200 isolates were duplicates from 100 isolates. Participating laboratories stored isolates at −70 °C until testing the antibiotic susceptibility using Etest strips. In addition to the test isolates, each laboratory received seven quality control N. gonorrhoeae strains, American Type Culture Collection (ATCC) 49226, F28, P681E, 10328, 10329, SPJ-15 and SPL-4 used in GISP [5].
Test procedure
At each laboratory, N. gonorrhoeae isolates were thawed sufficiently to remove a small amount for culture on chocolate agar. The plates were incubated at up to 24 h at 35–37 °C under an atmosphere containing 4–6 % CO2. A bacterial suspension was prepared and adjusted to a 0.5 McFarland standard for inoculating plates containing gonococcus (GC) agar base supplemented with either 1 % isovitalex (Becton Dickinson, Franklin Lakes, NJ, USA) or 1 % gonococcus haemophilus (GCHI) enrichment (Remel Laboratories, Lenexa, KS, USA). Azithromycin, cefixime and ceftriaxone Etest strips where placed on the agar surface in a manner consistent with the product insert. Following 18–24 h of incubation at 35–37 °C and 4–6% CO2, the MICs were determined by reading the lowest antibiotic concentration that inhibited growth. Collaborating laboratories used up to three different lots of Etest strips and between one to seven technicians to perform the testing.
Quality control
Quality control strains, with known MIC ranges determined by agar dilution, were included during each day of testing with the Etest (Table 1). The results of the quality control strains from the Etest MIC values for azithromycin, cefixime and ceftriaxone were compared to the established agar dilution MIC values and were within the established quality control range before validating the test run for analysis.
Table 1.
Quality control values for antimicrobial susceptibilities of reference strains of N. gonorrhoeae determined by agar dilution
| Antimicrobial agent | MIC (μg ml−1) range per N. gonorrhoeae strain | ||||||
|---|---|---|---|---|---|---|---|
| ATCC 49226 | F-28 | P681E | 10 328 | 10 329 | SPJ-15 | SPL-4 | |
| Azithromycin | 0.25–1.0 | 0.03–0.125 | 0.03–0.125 | 0.015–0.125 | 0.125–0.5 | 1.0–8.0 | 0.125–0.5 |
| Cefixime | 0.004–0.03 | 0.001–0.008 | 0.008–0.03 | 0.004–0.03 | 0.008–0.06 | 0.008–0.06 | 0.25–0.5 |
| Ceftriaxone | 0.004–0.016 | 0.001–0.002 | 0.002–0.008 | 0.002–0.015 | 0.004–0.03 | 0.004–0.015 | 0.03–0.25 |
Data analysis
Intra-laboratory variability of Etest MIC values within each laboratory was evaluated by analysis of the results from paired testing. The MIC values were rounded up to the nearest doubling dilution (log2) with essential agreement being within one log2 dilution.
For comparison with agar dilution MIC values, the Etest MIC values from the duplicate clinical isolates were rounded up to the nearest log2 dilution as suggested by the manufacturer. The percentage agreement was determined from Etest MIC values that were within one log2 dilution of the agar dilution MIC value.
RESULTS
Intra-laboratory variability
Seven of the laboratories tested all 200 isolates and one laboratory reported a single non-viable isolate which was excluded from the study to include the matched pair. The reproducibility of the Etest was 87.7 % for azithromycin, 93.2 % for cefixime and 95.6 % for ceftriaxone when assessed by essential agreement within one log2 dilution (Table 2). There were 5.5, 3.6 and 1.5 % errors of >two log2 dilutions difference for azithromycin, cefixime and ceftriaxone, respectively, among the eight laboratories. Two log2 dilution errors were slightly more common with azithromycin (6.6 %) than with cefixime (3 %) and ceftriaxone (2.9 %). There were no associations between the intra-laboratory variability of Etest MIC values and different Etest strip lots, the number of testing personnel or the use of either isovitalex or GCHI to supplement GC base agar.
Table 2.
Reproducibility of the Etest MIC values generated within each laboratory for the evaluation of N. gonorrhoeae
| Public health lab | No. of isolates tested in duplicate* | Azithromycin | Cefixime | Ceftriaxone | |||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Etest MIC† range | EA‡ | Two dilution error§ | Greater than two dilution error‖ | Etest MIC† range | EA‡ | Two dilution error§ | Greater than two dilution error‖ | Etest MIC† range | EA‡ | Two dilution error§ | Greater than two dilution error‖ | ||
| Lab 1 | 100 | 0.094–12.0 | 88 | 9 | 3 | 0.016–0.38 | 86 | 2 | 12 | 0.004–0.23 | 95 | 3 | 2 |
| Lab 2 | 100 | 0.047–6.0 | 88 | 5 | 7 | 0.016–0.25 | 92 | 0 | 8 | 0.002–0.19 | 99 | 1 | 0 |
| Lab 3 | 100 | 0.094–12.0 | 92 | 5 | 3 | 0.013–0.19 | 99 | 0 | 1 | 0.006–0.125 | 100 | 0 | 0 |
| Lab 4 | 100 | 0.032–4.0 | 86 | 8 | 6 | 0.023–0.75 | 97 | 2 | 1 | 0.004–0.25 | 99 | 1 | 0 |
| Lab 5 | 100 | 0.023–2.0 | 87 | 9 | 4 | 0.016–0.19 | 85 | 14 | 1 | 0.016–0.25 | 89 | 7 | 4 |
| Lab 6 | 100 | 0.038–12.0 | 84 | 8 | 8 | 0.016–0.16 | 91 | 4 | 5 | 0.016–0.16 | 92 | 7 | 1 |
| Lab 7 | 99 | 0.094–32.0 | 90 | 4 | 5 | 0.016–0.19 | 96 | 1 | 2 | 0.004–0.25 | 93 | 3 | 3 |
| Lab 8 | 100 | 0.025–12.0 | 86 | 6 | 8 | 0.016–0.25 | 99 | 1 | 0 | 0.047–0.19 | 97 | 1 | 2 |
| Total (%) | 799 | 701 (87.7) | 54 (6.6) | 44 (5.5) | 745 (93.2) | 24 (3) | 30 (3.6) | 764 (95.6) | 23 (2.9) | 12 (1.5) | |||
Laboratories were not aware that the isolates were duplicates.
MIC in ug ml−1.
Essential agreement interpreted as MIC values within one log2 dilution between duplicate isolates tested.
Two dilution error interpreted as MIC values (ug ml−1) that were two log2 dilutions different between duplicate isolates tested.
Greater than two dilution error interpreted as MIC values (ug ml−1) that were greater than two log2 dilutions different between duplicate isolates tested.
Comparison to agar dilution MIC
Overall, 90.7 % of the azithromycin Etest MIC values were within one log2 dilution of the agar dilution MIC values and considered to be in agreement with the agar dilution MIC (Table 3). Six of the eight participating laboratories had >90 % agreement between azithromycin Etest results and agar dilution MIC values. One laboratory reported results for 22 % of isolates that were <two log2 dilutions below the agar dilution MIC and 17.1 % of isolates tested in another laboratory were ≥two log2 dilutions above the agar dilution MIC. The overall agreement between Etest and agar dilution results was 74.3 % for cefixime (Table 4). Only two laboratories had >90 % agreement with cefixime. One laboratory had 16.5 % agreement with the majority, 83.5 %, of results being two or more log2 dilutions below the agar dilution result. A total of 1048 isolates (65.5 %) tested one or more log2 dilutions below the cefixime agar dilution MIC while only 70 (4.4 %) were one or more log2 dilutions above the agar dilution MIC. Agreement between the susceptibility test methodologies for ceftriaxone was 90.9 % and all eight laboratories had >80 % agreement (Table 5).
Table 3.
Summary of the MIC values determined by laboratories testing the azithromycin Etest to previously established agar dilution MIC values
| Public health laboratory | Agar dilution MIC range (μg ml−1)* | No. of isolates tested | Etest MIC values per isolate compared to agar dilution MIC (%) | ±1 Dilution from agar dilution MIC (%) No. of isolates | ||||
|---|---|---|---|---|---|---|---|---|
| ≥ −2 | −1 | 0 | 1 | ≥2 | ||||
| Lab 1 | 0.094–12.0 | 200 | 1 (0.5) | 43 (21.5) | 124 (62) | 24 (12) | 8 (4) | 191 (95.5) |
| Lab 2 | 0.047–6.0 | 200 | 17 (8.5) | 100 (50) | 74 (37) | 9 (4.5) | 0 (0) | 183 (91.5) |
| Lab 3 | 0.094–12.0 | 200 | 0 (0) | 53 (26.5) | 122 (61) | 21 (10.5) | 4 (2) | 196 (98) |
| Lab 4 | 0.032–4.0 | 200 | 6 (3) | 60 (30) | 113 (56.5) | 16 (8) | 5 (2.5) | 189 (94.5) |
| Lab 5 | 0.023–2.0 | 200 | 44 (22) | 86 (43) | 56 (28) | 14 (7) | 0 (0) | 156 (78) |
| Lab 6 | 0.038–12.0 | 200 | 5 (2.5) | 18 (9.1) | 83 (41.9) | 83 (41.9) | 11 (5.5) | 184 (92) |
| Lab 7 | 0.094–32.0 | 199 | 0 (0) | 5 (2.5) | 40 (20.1) | 120 (60.3) | 34 (17.1) | 165 (82.9) |
| Lab 8 | 0.047–12.0 | 200 | 11 (5.5) | 81 (40.5) | 94 (47.2) | 11 (5.5) | 3 (1.5) | 186 (93) |
| Totals | 1599 | 84 (5.3) | 446 (27.9) | 706 (44.2) | 298 (18.6) | 65 (4.1) | 1450 (90.7) | |
Agar dilution was performed at the Centers for Disease Control and Prevention.
Table 4.
Summary of the MIC values determined by laboratories testing the cefixime Etest to previously established agar dilution MIC values
| Clinical laboratory | Agar dilution MIC range (μg ml−1)* | No. of isolates tested | Etest MIC values per isolate compared to agar dilution MIC (%) | ±1 Dilution from agar dilution MIC (%) | ||||
|---|---|---|---|---|---|---|---|---|
| ≥ −2 | −1 | 0 | 1 | ≥2 | ||||
| Lab 1 | 0.016–0.38 | 200 | 16 (8) | 93 (46.5) | 78 (39) | 8 (4) | 5 (2.5) | 179 (89.5) |
| Lab 2 | 0.016–0.25 | 200 | 69 (34.5) | 80 (40) | 45 (22.5) | 5 (2.5) | 1 (0.5) | 130 (65) |
| Lab 3 | 0.013–0.19 | 200 | 14 (7) | 91 (45.5) | 85 (42.5) | 10 (5) | 0 (0) | 186 (93) |
| Lab 4 | 0.023–0.75 | 200 | 36 (18) | 107 (53.5) | 53 (26.5) | 2 (1) | 2 (1) | 162 (81) |
| Lab 5 | 0.016–0.19 | 200 | 35 (17.5) | 92 (46) | 63 (31.5) | 3 (1.5) | 7 (3.5) | 158 (79) |
| Lab 6 | 0.016–0.16 | 200 | 167 (83.5) | 24 (12) | 7 (3.5) | 2 (1) | 0 (0) | 33 (16.5) |
| Lab 7 | 0.016–0.19 | 199 | 43 (21.6) | 99 49.79.7 | 48 (24.1) | 4 (2) | 2 (1) | 151 (75.9) |
| Lab 8 | 0.016–0.25 | 200 | 11 (5.5) | 68 (34) | 102 (51) | 15 (7.5) | 4 (2) | 185 (92.5) |
| Totals | 1599 | 391 (24.5) | 657 (41.1) | 481 (30.1) | 49 (3.1) | 21 (1.3) | 1187 (74.2) | |
Agar dilution was performed at the Centers for Disease Control and Prevention.
Table 5.
Summary of the MIC values determined by laboratories testing the ceftriaxone Etest to previously established agar dilution MIC values
| Clinical laboratory | Agar dilution MIC range (μg ml−1)* | No. of isolates tested | Etest MIC values per isolate compared to agar dilution MIC (%) | ±1 Dilution from agar dilution MIC | ||||
|---|---|---|---|---|---|---|---|---|
| ≥ −2 | −1 | 0 | 1 | ≥2 | ||||
| Lab 1 | 0.004–0.23 | 200 | 1 (0.5) | 39 (19.5) | 118 (59) | 40 (20) | 2 (1) | 197 (98.5) |
| Lab 2 | 0.006–0.19 | 200 | 26 (13) | 86 (43) | 71 (35.5) | 15 (7.5) | 2 (1) | 172 (86) |
| Lab 3 | 0.006–0.125 | 200 | 0 (0) | 14 (7) | 119 (59.5) | 63 (31.5) | 4 (2) | 196 (98) |
| Lab 4 | 0.004–0.125 | 200 | 1 (0.5) | 42 (21) | 120 (60) | 35 (17.5) | 2 (1) | 197 (98.5) |
| Lab 5 | 0.016–0.25 | 200 | 1 (0.5) | 14 (7) | 65 (32.5) | 84 (42) | 35 (17.5) | 163 (81.5) |
| Lab 6 | 0.016–0.16 | 200 | 30 (15) | 77 (38.5) | 72 (36) | 19 (9.5) | 2 (1) | 168 (84) |
| Lab 7 | 0.06–0.25 | 199 | 2 (1) | 6 (3) | 81 (40.7) | 87 (43.7) | 23 (11.6) | 174 (87.4) |
| Lab 8 | 0.047–0.19 | 200 | 1 (0.5) | 21 (10.5) | 113 (56.5) | 54 (27) | 11 (5.5) | 188 (94) |
| Totals | 1599 | 61 (3.8) | 299 (18.7) | 758 (47.4) | 396 (24.8) | 82 (5.1) | 1453 (90.9) | |
Agar dilution was performed at the Centers for Disease Control and Prevention.
DISCUSSION
The evolving nature of antimicrobial resistance among N. gonorrhoeae requires laboratory support to perform antimicrobial susceptible testing of isolates from cases of suspect treatment failures to ensure proper patient management. An isolate, recovered from a patient following recommended treatment, which demonstrates high MICs to the administered therapy would alert disease control specialists to the potential emergence of a drug-resistant strain in the community. Agar dilution susceptibility testing is the reference method for determining antimicrobial susceptibilities of gonococcal isolates. However, this method is not well suited for most diagnostic laboratories because of the need to prepare a series of agar plates containing doubling dilutions of antibiotics. The agar plate dilution method is also tailored for high throughput processing since 25–35 isolates can be tested on one series of plates. In contrast, disk or strip diffusion methods may be preferred by laboratories that infrequently test isolates. Interpretive criteria which are required for understanding disk diffusion results have been established for antimicrobial activity against N. gonorrhoeae but does not include azithromycin and only has resistant classifications for ceftriaxone and cefixime [4, 14]. The Etest provides MIC values using a linear gradient and has been used for assessing antibiotic susceptibility in N. gonorrhoeae [16–20]. Some laboratories also use the Etest to generate data for reporting to the European Gonococcal Antimicrobial Surveillance Programme for trend analysis [24]. However, there has been no comprehensive studies on the performance of the Etest strips among laboratories testing N. gonorrhoeae for antibiotic susceptibility to azithromycin, cefixime and ceftriaxone.
In the present study, the essential agreement of the reproducibility of the Etest MIC values within each laboratory was >80 % for azithromycin, cefixime and ceftriaxone. The large number of errors that were ≥two log2 dilutions found with the reproducibility of azithromycin Etest results may be related to the MIC range of the isolates tested since azithromycin ranges were greater than either cefixime or ceftriaxone. The 12 errors with >two log2 dilutions difference that were observed from testing cefixime in one laboratory would not have altered the CLSI interpretive criteria of all isolates being susceptible [14]. However, another laboratory had one isolate with a cefixime Etest MIC value of 0.5 μg ml−1 and another with 0.75 μg ml−1, which were both above the CLSI susceptible breakpoint. The corresponding cefixime Etest MIC values from the replicate isolates were 0.094 and 0.25 μg ml−1, respectively.
The overall agreement, within one log2 dilution, between Etest and agar dilution MICs was >90% for azithromycin and ceftriaxone. Previous studies reported over 95 % essential agreement between Etest and agar dilution MIC values for ceftriaxone and 94% for azithromycin [20–22]. In contrast to the present data, approximately 20 % of ceftriaxone Etest MIC values were reported by Gose and colleagues [21] as being above the agar dilution suggesting that method-specific breakpoint values may be necessary to compare data between various methods. A larger number of comparative studies with a diverse bacterial population may be required to assess the performance of the ceftriaxone Etest with N. gonorrhoeae.
In our study, the majority of cefixime Etest MIC values were lower than the agar dilution MIC values which resulted in slightly <80% overall agreement. Three laboratories had approximately 90% agreement while one laboratory had only 16.5 % agreement. Given that the poor agreement was isolated and not attributable differences due to Etest lots or methodology, training may improve the performance. Cefixime Etest MIC values may also be underreported as compared to MIC values from the agar dilution method as previously suggested [23]. The tendency for low Etest cefixime MIC values compared to agar dilution may require adjustments to interpretive breakpoints.
A limitation of this study was the inability to test a wider range of N. gonorrhoeae isolates with elevated MIC values to ceftriaxone and cefixime since clinical isolates are rare. Testing clinical isolates with higher MIC values would provide a more detailed analysis but such isolates have yet to be identified in the United States. In the absence of comparative data with elevated MIC values to ceftriaxone and cefixime, it is recommended that isolates with these values should be tested by agar dilution for confirmation.
In conclusion, these data indicate that the Etest can reliably detect emerging N. gonorrhoeae resistance to front-line antibiotics with results that are comparable to agar dilution. The poor intra-laboratory correlation by some participating laboratories would likely be corrected with training and experience. Additional studies testing clinical N. gonorrhoeae isolates with a wider range of higher MIC values, especially ceftriaxone and cefixime, should be conducted to determine if the correlation between Etest and agar dilutions is maintained.
Acknowledgements
The authors thank excellent technical support provided by Kevin Pet-tus, Samera Bowers, Christi Philips, Carol Farshy, Sarah Buss PhD and Amity Roberts PhD. Comments from Jean Patel, Chris Whelen, Chaney Waters and Paula Snippes Vagnone were greatly appreciated.
Funding information
This research was supported under Cooperative Agreement U60HM000803 between the Association of Public Health Laboratories (APHL) and the Centers for Disease Control and Prevention. Marie-Claire Rowlinson, Norman P. O’Connor, Jason Wholehan, Jafar H Razeq, Anita Glennen, Dapne Ware, Peter C. Iwen and Lillian V. Lee received funding from the APHL under Cooperative Agreement U60HM000803. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the Centers for Disease Control and Prevention.
Abbreviations:
- ATCC
American Type Culture Collection
- CDC
Centers for Disease Control and Prevention
- CLSI
Clinical Laboratory Standards Institute
- GC
gonococcus
- GCHI
gonococcus haemophilus
- GISP
Gonococcal Isolate Surveillance Project
Footnotes
Conflicts of interest
The authors declare that there are no conflicts of interest.
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