The Diagnostic Capacity of Three Phenotypic Techniques of Extended-Spectrum β -Lactamase Detection

The of AbstractBackground: Early detection of extended-spectrum β -lactamases (ESBLs) producing bacteria is critical for infection prevention and control. Numerous phenotypic approaches and automated systems have been developed for detecting ESBL bacteria. However, there is a scarcity of data in Ethiopia regarding the most reliable, simple, and cost-effective methods for detecting ESBL-producing bacteria. This study, therefore, aimed to evaluate the diagnostic performance of three phenotypic approaches for detecting ESBL-producing bacteria. Methods: In this study, 117 isolates of Klebsiella pneumoniae , Escherichia coli , Klebsiella oxytoca , and Proteus mirabilis were examined. Cefotaxime (30 µg) and ceftazidime (30 µg) were used for screening ESBL enzymes. A screening breakpoints of ≤ 27 mm and ≤ 22 mm were used for cefotaxime (30 µg) and ceftazidime (30 µg), respectively, as per the Clinical and Laboratory Standards Institute (CLSI) guidelines. All 117 strains were further confirmed by the Vitek 2 compact, double disk synergy, ESBL Epsilometer test, and combined disk method. The combined disk method was adopted as the reference method. Results: Out of 117 isolates, 90 (86%) had zone diameters of ≤ 27 mm and ≤ 22 mm for cefotaxime (30 µg) and ceftazidime (30 µg), respectively. The reference method detected 76 (65%) ESBL isolates out of 117 ones. From among the three techniques (i.e., double disk synergy, Vitek 2 compact, and ESBL Epsilometer test), the double disk synergy method demonstrated overall sensitivity and specificity of 97.4% and 97.6%, respectively. Vitek-2, cefotaxime, and ceftazidime Epsilometer test indicated indeterminate results of 6.8%, 6.8%, and 5.1% respectively. Conclusion : Double disk synergy was found to have the highest sensitivity and specificity for detecting ESBL isolates with no indeterminate results.

and simple testing techniques for detecting these enzymes in Enterobacterales isolates for epidemiological purposes. In Ethiopia, there is a scarcity of data regarding the most reliable, uncomplicated, and cost-effective techniques for the laboratory detection of extended-spectrum and lactamase-producing bacteria. As a result, the current study aimed to evaluate the diagnostic accuracy of three phenotypic approaches for detecting ESBL-producing bacteria.

Study Design, Area, and Period
This cross-sectional study was conducted from January 5 to June 30, 2020, at the National Clinical Bacteriology and Mycology Reference Laboratory of Ethiopian Public Health Institute, in which Klebsiella pneumoniae, Escherichia coli, Klebsiella oxytoca, and Proteus mirabilis isolates recovered from clinical specimens were examined.

Sample Size Determination
The CLSI EP09-A3 recommended using at least 40 and preferably 100 samples to compare two laboratory methods. Therefore, a total of 117 above-mentioned isolates were collected in order to compare laboratory methods of ESBL detection (14).

Bacterial Culture and Identification
During the study period, 265 clinical samples were collected. In the event of sample transit delays, appropriate transport media were used. The isolates used in this investigation were recovered from blood, bodily fluids, wounds, sputum, and urine specimens. All specimens were inoculated onto appropriate culture media and cultured at proper temperatures and incubation time according to the per laboratory standard operating protocols (15). The isolates were identified by performing traditional biochemical tests: triple sugar iron agar (Liofilchem, Italy), oxidase strips (Liofilchem, Italy), Simon's citrate agar (Liofilchem, Italy), and lysine iron agar (Liofilchem, Italy). Indole production and motility using sulfide-indolemotility medium (Liofilchem, Italy). Urease production using a urea agar base supplemented with 40% urea solution (Oxoid Ltd., England) (15). Also, 117 isolates of E. coli, K. pneumoniae, P. mirabilis, K. oxytoca isolates were isolated during the study period ( Figure 1).

Double-disk Synergy Test (DDST)
Third-generation cephalosporins (cefotaxime and ceftazidime) were applied along with a third-generation cephalosporins disk containing clavulanic acid (amoxicillin-clavulanic acid) (Oxoid Ltd., England) (Oxoid Ltd., England). Enhanced inhibition zones around either of the disks in the direction of the clavulanic acid disk were considered ESBL positive. As for cephalosporin 30 µg disks, the gap between the disks was 20mm from center to center (6) (Figures 2 and 3).

VITEK 2 System (bioMérieux)
VITEK 2 system antimicrobial susceptibility testing cards were used for doing (AST-GN86) (bioMérieux, Durham, North Carolina, USA). The results were analyzed using Vitek-2 compact software version 7.0, and The result was interpreted by the Vitek-2 compact software knowledge data base called the advanced expert system, which is used to interpret the antimicrobial susceptibility testing results. (17).

Quality Control
Klebsiella pneumoniae ATCC 700 603 and E. coli ATCC 25 922 Standard American type culture collection strains were used as quality control strains.

Statistical Analysis
The detection power of each method was evaluated based on its sensitivity, specificity, and positive and negative  (Table 1).

ESBL Detection Using E-test
The ESBL E-test ceftazidime/ceftazidime + clavulanic acid strips showed sensitivity, specificity, positive predictive   (Tables 2  and 3). The ESBL E-tests showed a very good measure of agreement with the reference method (kappa value = 0.827-0.877, P value = 0.001) (Tables 2 and 3).

Discussion
The new extended-spectrum cephalosporin and aztreonam breakpoints reduced the possibility of determining ESBL producers susceptible to these antibiotics. However, timely detection of ESBL-producing Enterobacterales is also very important for early designing and implementing control strategies (1,2).

Vitek 2 Compact System
In our study, the Vitek 2 compact method demonstrated a sensitivity and specificity of 88.2% and 82.9%, respectively. This result was in line with the findings from Wiegand et al study in which the sensitivity and specificity were 86% and 78%, respectively (13), and from Thomson et al study in which the sensitivity and specificity were 89% and 85%, respectively (18

Double-Disk Synergy Methods
In this study, the overall sensitivity and specificity of the DDST were detected to be 97.37% and 97.56%, respectively. Our findings are partially consistent with the findings of other researchers who investigated the diagnostic capacity of double disk synergy methods using ESBL and non-ESBL producing bacteria (ranging from 79% to 97% and 94% to 100%, respectively), which were initially confirmed using PCR methods (11,23,24,31,32). However, our study findings contradicted the results from the studies by Yang et al reporting sensitivity of 96.0% and specificity of 69.2% for the double-disk synergy method (25), and by De Gheldre et al determining sensitivity of 89% and specificity of 92% (33). The observed inconsistency was likely due to the different Enterobacterales strains used (in this study, only K. pneumoniae, K. oxytoca, P. mirabilis, and E. coli were used, whereas K. aerogenes, P. vulgaris, Providencia stuartii, and others were applied in the studies by other researchers), regional differences, and so on.

Conclusion
In sum, Vitek-2 compact and ESBL E cefotaxime/ cefotaxime with clavulanic acid yielded inconclusive findings of 6.8 percent. In addition, ESBL E ceftazidime/ ceftazidime with clavulanic acid produced 5.1 percent indeterminate findings. However, the double-disk synergy method was shown to detect more ESBL producers with no indeterminate results. Therefore, it was concluded that the double-disk synergy method was more reliable, simpler, and less expensive method which also required no specialized equipment or considerable expertise.

Ethical Approval
The Ethical clearance of this study was obtained from the Ethical Review Committee of the Department of Medical Laboratory Sciences, College of Health Sciences, Addis Ababa University (DRERC/474/19/MLS). In addition, the Ethiopian Public Health Institute granted an official permission.