November 2003, Volume 53, Issue 11

Original Article

Comparison of Double Disc and Combined Disc Method for the detection of Extended Spectrum ß Lactamases in Enterobacteriaceae

K. Jabeen  ( Department of Pathology, Aga Khan University, Karachi. )
A. Zafar  ( Department of Pathology, Aga Khan University, Karachi. )
R. Hasan  ( Department of Pathology, Aga Khan University, Karachi. )

Introduction

Extended-spectrum ß-Lactamase (ESBL) producing organisms are a major problem in the area of infectious disease1 after discovery in 1983.2,3 These ß-Lactamases can be produced by a variety of Enterobacteriaceae; however, the most common ESBL-producing organisms are Klebsiella species, and Escherichia coli.4,5 These organisms confer resistant to all ß-Lactam antibiotics except cephamycins and carbapenems.4,6,7 In addition, ESBL-producing organisms are frequently resistant to many other classes of antibiotics; including aminoglycosides and fluoroquinolones4,8 thus treatment of these infections is often a therapeutic challenge. ESBL arise by mutations in genes of plasmid mediated beta lactamases (especially TEM and SHV enzymes).8 A second type of transmissible resistance arises from plasmid acquisition of a chromosomal amp C gene providing resistance to cephamycins as well.9
The frequency of ESBL-producing organisms differs significantly by geographic location.10-12 The SENTRY surveillance program reported the frequency of ESBL-producing K. pneumoniae as 45% in Latin America versus 7% in the United States.10 Although considerable variation exists in the U.S., with a surveillance study of 15 hospitals in Brooklyn, New York, reporting that 34% of their K. pneumoniae produce ESBL.11 Frequency of ESBL producing E. coli at hospitals in the SENTRY surveillance program was much lower, 1-8%.10 In Pakistan, a study performed in Islamabad shows a prevalence of 48% in nosocomial isolates.13 Another study from Rawalpindi reported 35% ESBL detection in nosocomial isolates.14
Detection of ESBL is challenging for the clinical microbiology laboratory.15 Its presence in the bacterial cell does not always produce a resistant phenotype; some ESBL isolates may appear susceptible to third-generation cephalosporins in vitro. Failure of either MIC or disc test alone to accurately detect the presence of an ESBL has been well documented.16,17 A proficiency testing project for clinical laboratories participating in the National Nosocomial Infections Surveillance System indicated that as many as 58% of laboratories failed to detect and report ESBL isolates correctly.18 In some studies, 37% of ESBL producing organisms were misreported19 whereas in another study only 7 out of 38 laboratories correctly identified these organisms.20 These data suggest that improvements in the ability of clinical laboratories to detect ESBLs are needed as all ESBL producing organisms should be reported as being resistant to all penicillins, cephalosporins and aztreonam.21
National Committee for Clinical Laboratory Standards (NCCLS) and British Society for Antimicrobial and Chemotherapy (BSAC) have made a standardized criterion for screening, confirmatory testing and reporting for these organisms.21,22 Several ESBL detection test have been proposed based on Kirby Bauer disc diffusion methodology such as the double disc approximation test23 combined disc method22 and three-dimensional test.24 In addition, MIC based methods in which dilution tests are performed.25 Several commercial tests (Etest ESBL strips) as well as automated systems (Vitek) are also available for detection of ESBL.26 None of the tests for phenotypic detection of ESBL are 100% sensitive or specific, however double disc approximation test, combined disc method and broth dilution MIC method are the easiest and cost effective methods for use by many clinical laboratories.26
Detection of ESBL was initially performed by double disc approximation test in our laboratory, however there were person-to-person variation in the interpretation of results. We therefore, conducted a study comparing double disc approximation and combined disc method for the detection of ESBL.

Material and Methods

The study was done in a 550-bed tertiary care hospital located in Karachi, Pakistan. The clinical microbiology laboratory receives samples of patients presenting to the hospital as well as outside referrals from other hospitals, clinics and general practitioners across the city.
In this comparative study, samples were taken of patients seen at the AKUH, or those referred from other hospitals, clinics and general practitioners across the city.

Laboratory Methods

All urinary specimens growing members of the family Enterobacteriaceae. These isolates were identified by routine biochemical tests.27 Disc susceptibility tests were performed by criteria according to NCCLS21 using Mueller Hinton agar (Oxoid). ESBL detection was done by combined disc method using cefotaxime (30ug) versus cefotaxime plus clavulanate (30+10 ug) (Oxoid) according to NCCLS criteria.21 Double disc method was performed by using disc containing co-amoxiclavulanic acid (20+10 ug) and aztreonam applied 10 mm apart.22
Data analysis was done using SPSS version 10. Frequencies were calculated by using descriptive statistics and expressed as percentages and rate.

Results

During the study period 471 isolates were identified as Enterobacteriaceae. Frequent isolates were E.coli (74%), K. pneumoniae(13%), Enterobacter sp. (9%), Proteus sp. (3%). ESBL production was detected in 140 (30%) isolates by combined disc method and 139 (29.5%) by double disc method. No significant difference was found between the two methods. ESBL production by the organisms studied is shown in Figure 1 exhibiting that frequency of ESPL production was highest in Enterobacter with 34% of 41 Enterobacter tested being ESBL positive. Of the ESBL positive isolates, 41/140(29%) gave zone diameters with cefuroxime, cefixime, cefotaxime and aztreonam that were within the sensitivity range cutoff and would have been falsely reported as being beta lactam sensitive in absence of ESBL detection (Figure 2).

Discussion

Detection of ESBL is a major challenge for the clinical microbiology laboratory15 as its detection has major impact on therapy. Moreover, presence of an ESBL also has significant infection control implications.26
We have compared two methods for ESBL detection based on Kirby Bauer disc diffusion methodology. Both of these methods were found to be equivalent in detection of ESBL producing isolates. However in the double disc approximation method distance of disc placement is not standardized and it has been suggested that reducing the distance between two discs could increase sensitivity of this method.23 Combined disc method is a recently developed method with ability to detect presence of ESBL.28 A study reported by Zali et al29 have documented sensitivity of double discs with ceftazidime plus clauvulanic acid as being 86% and with cefotaxime plus clauvulanic acid as being 65% and recommended use of both ceftazidime and cefotaxime combinations to increase the sensitivity upto 93%.29
Our results show that in the absence of ESBL detection 29% of isolates would have been reported as being falsely sensitive with cefuroxime, cefixime, cefotaxime and aztreonam. Livermore et all have reported that 37% of ESBL producing organisms were misreported in their study.19 A study conducted by World Health Organization using disc diffusion technique found that 5.4% of laboratories found an ESBL producing strain to be susceptible to all cephalosporins.30
Double disc method needs two discs which are part of routine sensitivity in most laboratories, whereas combined disc method needs only one extra disc in the regular testing battery. Therefore expense of determination of ESBL is minimal. However as use of double disc method needs accurate distance between two discs so there is a need of precision and person to person variation could occur.
We therefore, recommend that determination of ESBL production is necessary to prevent misreporting and hence treatment failure. This detection could be done by either of the two methods, as both of these methods have found to be simple as well as cost effective.

Acknowledgements

We are thankful to all the technical staff of the clinical microbiology for their help and support.

References

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Abstract

Objective: To compare double disc approximation and combined disc method for their ability to detect extended spectrum b lactamase (ESBL) production in enterobacteriaceae and determine the percentage of isolates which are falsely reported as sensitive in absence of ESBL detection, in a clinical microbiology laboratory of a tertiary care hospital between September - October 2002.

Methods: Selected isolates were identified according to standard biochemical tests. Disc susceptibility tests were performed according to NCCLS. ESBL detection by combined disc {cefotaxime (30ug) versus cefotaxime plus clavulanate (30+10 ug)} was compared with detection using double discs {amoxy-clavulanic acid (20+10 ug) and aztreonam (30ug) applied 10 mm apart}. Results were interpreted according to NCCLS and analysed on SPSS version 10.

Results:
ESBL production was detected in 140 (30%) isolates by combined disc method and 139 (29.5%) by double disc method. There was no significant difference between two methods. Of the ESBL positive isolates 41(29%) gave zone diameters that were within the sensitivity range cutoff and would have been falsely reported as being beta lactam sensitive in absence of ESBL detection.

Conclusion: ESBL detection should be routinely performed in clinical laboratories as false reporting would result in treatment failure despite in vitro sensitivity. No difference was found between the combined disc and double disc methods hence either of two could be used (JPMA 53:534;2003).

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