a Antibiotic Resistance Monitoring and Reference Laboratory, b Laboratory of Enteric Pathogens, Central Public Health Laboratory, c Communicable Disease Surveillance Centre and d Statistics Unit, Public Health Laboratory Service, 61 Colindale Avenue, London NW9 5HT, UK
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Abstract |
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Introduction |
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To validate the quality of routine data for one important pathogen, we compared the resistance rates recorded for Escherichia coli from blood and cerebrospinal fluid (CSF) based on (i) routine data reported to the Public Health Laboratory Service (PHLS) by hospitals in England and Wales and (ii) results for isolates tested by the Laboratory of Enteric Pathogens (LEP) at the Central Public Health Laboratory. E. coli was chosen as the most common cause of bacteraemia in the UK2 and because of data availability. Some parts of each data set have been published previously, but without cross-validation.3,4
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Materials and methods |
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Since 1989, up to 220 hospitals in England and Wales have voluntarily reported antibiotic susceptibility data for isolates from blood and CSF to the PHLS-CDSC.4,5 Most reporting was initially on paper forms but, since 1994, there has been a progressive Region-by-Region switch to CoSurv, an electronic reporting system developed by the PHLS.6 Reports with antibiotic susceptibility information were downloaded from the LabBase database5,6 for analysis.
UK hospitals perform susceptibility tests by their own choice of technique. Most (>90%) use some variant of Stokes' comparative method, but minorities use Kirby Bauer-type disc tests with the control strain on a separate plate, or use breakpoint testing.7 The choice of media, supplements and the method of inoculum preparation varies between sites, but most laboratories use IsoSensitest or DST agars with semi-confluent bacterial lawns.8 The antibiotics tested likewise are at the discretion of the laboratories, and reporting is variable, so that up to 45% of CoSurv/ LabBase reports for E. coli bacteraemias have no susceptibility data attached.
LabBase data for ampicillin and amoxycillin were pooled for analysis. Isolates reported as intermediate to any drug were counted as resistant, since many laboratories do not distinguish these categories. All the hospitals that contribute data participate in the National External Quality Assurance Scheme (NEQAS) and the results of this system provide the primary quality assurance. Among 13 NEQAS distributions of Enterobacteriaceae in the study period, the percentage of laboratories submitting correct results ranged from 97.4 to 100% (mean 99.3%) for ampicillin and from 92.6 to 99.5% (mean 96.9%) for trimethoprim (J. Snell, personal communication).
Susceptibility testing of E. coli isolates referred to LEP
From 1991 to 1997, LEP received 1526 E. coli isolates from bacteraemic patients. All were identified, serotyped and tested for resistance to a panel of antimicrobial drugs.3 Susceptibility testing was undertaken for surveillance purposes and the results are not communicated to the laboratories that sent the isolates, meaning that there was no incentive to send isolates with unusual resistances. These isolates were subjected to breakpoint tests with various antimicrobials, including ampicillin, 8 mg/L; ciprofloxacin, 0.125 and 1 mg/L; gentamicin, 4 mg/L and trimethoprim, 2 mg/L. MIC determinations with wide ranges of dilutions were undertaken for random selections of the isolates found to be resistant at these breakpoints. The medium for MIC and breakpoint tests was IsoSensitest agar (Oxoid, Basingstoke, UK); the inoculum was 104 cfu/spot; and results were read after 18 h at 37°C, with thin hazes counted as no growth. E. coli NCTC 10418 was used as a susceptible control and was tested in parallel.
Statistical analyses
The annual trends in the proportions of isolates resistant to each antibiotic in the LabBase and LEP data sets were determined by logistic regression.9 The difference between the proportion resistant in each data set and the 95% confidence intervals (CI) for these differences were determined by standard methods.10
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Results |
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Figure 2 re-plots the LabBase and LEP data against time, indicating also the highest and lowest proportions of resistant E. coli from any Regional Health Authority (RHA, based on pre-1994 boundaries) in each year, as based on the LabBase data. The RHAs with the lowest and highest resistance rates varied from year to year but East Anglian and South Western consistently had the smallest proportions of ampicillin- and trimethoprim-resistant isolates (PHLS data on file). Except for ciprofloxacin, the proportion of resistant isolates found by LEP fell within the ranges bounded by the highest and lowest rates for individual RHAs. In the case of ciprofloxacin, the proportion of isolates recorded by LEP was marginally above the highest rate for any RHA in 6 of the 7 years considered.
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Detection of resistance by routine laboratories is most likely to be reliable where this resistance is high level. To examine this factor, groups of the isolates found resistant in LEP's breakpoint tests were subjected to MIC testing with full ranges of doubling dilutions of antibiotics (Table II). MICs of ampicillin, trimethoprim and gentamicin for resistant isolates were mostly (>97% of cases) at least eight-fold above the breakpoints, whereas much (27%) of the resistance to ciprofloxacin was low level (MICs 0.251 mg/L) and isolates with high level resistance (MIC > 1 mg/L) only began to dominate after 1995.
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Discussion |
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E. coli was considered as it is the most common isolate from bacteraemic patients2 and because complementary (though unpaired) data sets were available. Both data sets indicated significantly increasing proportions of isolates resistant to trimethoprim, ciprofloxacin and ampicillin, and suggested a slow erosion in the proportion of isolates susceptible to gentamicin. The proportions of resistant isolates recorded in the two data sets were also generally similar, with the 95% CI on their differences overlapping zero in 4 or 5 of the 7 years for each antibiotic (Figure 1). Nevertheless, (i) the LEP results indicated a much sharper rise in ampicillin resistance than the LabBase data, (ii) the LEP results indicated lower rates of gentamicin resistance than the LabBase data and (iii) the LabBase data indicated lower rates of resistance to ciprofloxacin than the LEP data. Such disagreement may reflect bias in the sample of isolates received by LEP or reported into LabBase. Bias in the LEP sample is clearly possible since this group comprised only 166286 isolates per annum from a total of 63 laboratories, and with the isolates selected by the submitting laboratories. Some laboratories submit all E. coli isolates from bacteraemic patients; others never submit such isolates. The plots in Figure 2
aim to control for this potential bias by illustrating the extent of geographical variation in the LabBase data. The rationale is that sampling bias would be unlikely to push the proportions of resistant isolates received by LEP beyond the ranges bounded by the highest and lowest proportions reported for individual RHAs. This prediction was fulfilled: the LEP data lay outside these bounds only for ciprofloxacin, a drug where there is particular scope for laboratory-to-laboratory variation in the definition of resistance (see below). Potential sources of geographical bias also can be identified in the LabBase data: London teaching hospitals were under-represented for much of the survey period and no susceptibility data are attached to nearly half the reports of E. coli bacteraemia received. It is unclear whether these omissions are randomly distributed with respect to resistance. A bias towards submission of resistant isolates to LEP is unlikely as these organisms were sent for serotyping and, since susceptibility data are not reported, there is no incentive to submit resistant isolates. Distortion of the LEP data by multiple inclusion of epidemic strains can be discounted since serotyping indicated wide diversity.3
Besides sampling bias, differences in categorization of isolates as resistant or susceptible are the other major potential source of variation. Such differences may reflect non-standardized methodology among routine laboratories or different definitions of resistance. LEP used breakpoints as follows: ampicillin, 8 mg/L; ciprofloxacin, 0.125 mg/L; gentamicin 4 mg/L and trimethoprim 2 mg/L; most clinical laboratories use Stokes' plates with resistance (or intermediate resistance) counted as a zone radius more than 3 mm smaller than for the E. coli NCTC 10418 control.7,8 These differences are unlikely to have distorted apparent resistance rates to ampicillin or trimethoprim, where most resistance in E. coli is high level (Table II), but may have affected the comparisons for gentamicin and ciprofloxacin. The LEP breakpoint for gentamicin is 16-fold above the MIC for E. coli NCTC 104188 and isolates with MICs below this breakpoint might be expected to give zones more than 3 mm smaller than for this control, meaning that they would be reported as resistant in LabBase. This may explain why LabBase data indicated higher rates of gentamicin resistance than LEP data. Ciprofloxacin is more complex because resistance accrues stepwise and is often low level (Table II
), meaning that small differences in the definition of resistance can radically change the proportion of isolates classed as resistant. LEP ciprofloxacin data were analysed primarily against a low breakpoint of 0.12 mg/L, aiming to identify isolates with any degree of reduced susceptibility. The desirability of screening for such reduced susceptibility to ciprofloxacin in Enterobacteriaceae has been highlighted recently.11 Probably as a result of this low breakpoint, the proportion of resistant isolates found by LEP was higher than that recorded in any individual RHA in most years (Figure 2
). Application of a ciprofloxacin breakpoint of 1 mg/L (as advocated by the BSAC8 and as used by LEP as a high breakpoint3) puts the trend line for the LEP data between the highest and lowest RHA rates in Figure 2d
. A further complicating factor is that some routine laboratories follow the BSAC's 1991 advice to define ciprofloxacin resistance as an annular zone radius more than 7 mm smaller than the E. coli NCTC 10418 in Stokes' tests,8 whereas others define resistance as a zone more than 3 mm smaller than this control, as with other antimicrobials. Even discounting variation in test performance, these differences in definition mean that it is scarcely surprising that the rates of resistance recorded by LEP and the routine data diverge: what is more critical is that both data sets indicated rising trends in resistance (Table I
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The general agreement between the LabBase and LEP data confirms that resistance to ampicillin/amoxycillin has long been frequent among E. coli in the UK; that resistance to trimethoprim has risen from c. 20% in 1991 to 30% in 1997; that ciprofloxacin resistance, virtually unknown in E. coli in 1991, is now seen in 35% of isolates and that gentamicin resistance has increased but remains extremely rare. The agreement supports the view that the routine data are sufficiently robust to be related to prescribing and population data, despite the diverse methodologies used in the study period. This latter situation should improve further with the standardization of routine tests, now coming under a BSAC initiative. It should be stressed that these conclusions apply only for the present combinations of antibiotics and organism. Co-amoxiclav and cefuroxime are widely used against E. coli infections but were not considered here because they were not tested by LEP for much of the period 19911997; they may present problems since their breakpoints are only slightly above the modal MICs for the species and because much resistance is low level. Other bacteria need case-by-case validation. In the case of Pseudomonas aeruginosa we found poor agreement between routine and central data sets for aminoglycosides, ß-lactams and quinolones.12 Finally, it should be emphasized that the present analysis was unpaired and did not entail comparison of routine and in-house results for individual isolates, only overall rates, and many individual disagreements may be hidden.
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Notes |
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References |
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9 . Clayton, D. & Hills, M. (1993). Poisson and logistic regression. In Statistical Models in Epidemiology, pp. 22736. Oxford University Press, Oxford.
10 . Bland, M. (1991). Introduction to Medical Statistics, pp. 1423. Oxford University Press, Oxford.
11 . Murphy, O. M., Marshall, C., Stewart, D. & Freeman, R. (1997). Ciprofloxacin-resistant Enterobacteriaceae. Lancet 349, 10289.
12 . Livermore, D. M. & Chen, H. Y. (1999). Quality of antimicrobial susceptibility testing in the UK: a Pseudomonas aeruginosa survey revisited. Journal of Antimicrobial Chemotherapy 43, S1722.
Received 29 January 1999; returned 29 April 1999; revised 24 June 1999; accepted 8 October 1999