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ORIGINAL PAPER
Standardised procedures can improve the validity of susceptibility testing of uropathogenic bacteria in general practice Lars Bjerrum1,2, Per Grinsted 3, Per Hyltoft Petersen4 and Per Søgaard 5 Research Unit of General Practice, University of Southern Denmark, Odense, Denmark Department of Clinical Pharmacology, University of Southern Denmark, Odense, Denmark 3 General Practice Liaison Programme, Odense University Hospital, Odense, Denmark 4 Department of Clinical Biochemistry, Odense University Hospital, Odense, Denmark 5 Department of Clinical Microbiology, Odense University Hospital, Odense, Denmark 1 2
Scand J Prim Health Care 2000;18:242 – 246. ISSN 0281-343 2 Objective – To investigate whether the validity of susceptibility testing in general practice would improve when preceded by an intervention. Intervention – Instruction in standardised susceptibility testing procedures given by laboratory instructors. Method – Urine specimens containing monocultures of typical uropathogenic bacteria were sent to 23 general practices before and after the intervention. Practices performed susceptibility testing by the Sensicult and the Iso-Res agar methods and the validity of the results before and after the intervention was compared. Results from susceptibility testing at the bacteriological laboratory, Odense Uni-
Urinary tract infections (UTI) are responsible for about 2 – 5% of contacts in general practice. The diagnosis is usually based on the demonstration of signicant bacteriuria by stix, phase-contrast microscopy or semi-quantitative culture of a midstream specimen of urine (1). If required, the choice of antibiotic treatment can be based on susceptibility testing performed in general practice (2,3). Susceptibility testing directly from the urine specimen is easy to perform, rapid and economical for the general practitioner (4 – 10). However, studies of the validity of susceptibility testing in general practice suggest a lower validity compared to results obtained from bacteriological laboratories (3,11 – 13). In an earlier, multipractice, study we found more than 10% false results and that prediction of resistance, in particular, was inadequate (11). We found only a few studies evaluating the effect of training in susceptibility testing in general practice and the results are inconsistent (2,7). The aim of this study was to investigate whether introduction of a set of standardised procedures targeting two different susceptibility methods (Sensicult and Iso-Res agar) could improve the validity of susceptibility testing. Before performing the study we agreed on an acceptable level of quality if the prevalence of false results was lower than 5%. Scand J Prim Health Care 2000; 18
versity Hospital, were used as gold standard. Results – The median frequency of correct results increased from 82% to 98% for susceptibility testing based on Sensicult (p 0.001) and from 90% to 96% based on Iso-Res agar (p 0.05). Conclusion – The validity of susceptibility testing in general practice improves when preceded by instruction in standardised procedures. Key words: microbial sensitivity tests, urinary tract infections, teaching, family practice, test validity, standardised technique. Lars Bjerrum, Research Unit of General Practice, University of Southern Denmark, Winsløwparken 19, DK-5000 Odense, Denmark. E-mail: l-bjerrum@ health.sdu.dk
MATERIAL AND METHODS Selection of practices, antibiotic susceptibility testing and intervention The study was carried out during 1995 and 1996 as a multipractice investigation in the County of Funen, Denmark (467 000 inhabitants, 174 general practices). A random sample of 25 practices was asked to participate, and 23 agreed. Before the intervention was launched, all practices examined a set of urine specimens (see below) by two different susceptibility tests: a dip-slide system consisting of a 3*9 cm2 dip-slide, on both sides covered with a modied Mueller-Hinton agar (Sensicult, Orion Diagnostica) with application of Biodisk (Solna, Sweden) and a conventional agar plate system consisting of a 9 cm plate with Mueller-Hinton agar (Iso-Res agar, Becton-Dickinson) with application of Neo-Sensitabs (ROSCO). The intervention consisted of careful instruction in standardised susceptibility testing procedures, given by mobile laboratory instructors. Each practice was visited two times and instructed in the procedures as shown in Fig. 1. The time used for a visit varied from practice to practice and ranged from 1 to 3 hours. Shortly after the intervention was launched a new set of urine specimens was distributed to the same practices. Results of both types of susceptibility test (Sensicult and Iso-Res agar) before and after the
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intervention were compared with results from a gold standard. The results of a standardised culture method performed by skilled bacteriologists at the bacteriological laboratory were used as the gold standard; these have been shown to have a high validity (14). For each practice, the percentage of correct results after intervention was compared with the percentage of correct results before intervention. Urine specimens Urine specimens were created at the bacteriological laboratory of Odense University Hospital by adding an approximately known quantity of typically uropathogenic bacteria to a sterile urine specimen. The cultures employed were Escherichia coli, Klebsiella pneumonia, Proteus mirabilis, Enterobacter cloacae and Enterococcus faecalis. The strains were
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obtained from patients with UTI and the pattern of susceptibility corresponded to the occurrence of resistant bacteria among patients in general practice. Overnight cultures were diluted and a suitable volume was transferred to a tube with 10 ml of sterile urine yielding a bacterial concentration of 10 000 to 10 000 000 bacteria per millilitre. Specimens were transported refrigerated to the GP surgery and susceptibility testing was performed the same day the specimens were received. Each practice examined 28 urine specimens, 12 before the intervention and 16 after the intervention. Urine specimens to be tested at the bacteriological laboratory (gold standard) were the same as those sent to the GP surgeries, and they were handled in precisely the same way (transported refrigerated) for practices and the bacteriological laboratory.
Fig. 1. Standardised procedures for susceptibility testing in general practice. Scand J Prim Health Care 2000; 18
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Table I. Percentage of correct results of susceptibility testing before and after intervention according to the type of antibiotic examined. Two sets of urine specimens (12 before the intervention and 16 after the intervention) were analysed in 23 general practices (644 analyses). Susceptible bacteria Before intervention Sulfamethizol Ampicillin Mecilinam Nitrofurantoin Trimetroprim Ooxacin
60.6 73.0 76.5 95.0 80.6 95.7
(52.4–68.8) (65.6–80.4) (64.8–88.1) (91.0–98.9) (71.4–89.7) (92.0–99.4)
After intervention 94.3 68.3 96.6 96.1 99.0 96.1
(90.8–97.9) (61.0–75.1) (93.9–99.2) (93.0–99.1) (97.9–100) (93.9–98.3)
RESULTS Table I gives the percentage of correct results of susceptibility tests before and after the intervention according to the different antibiotics assessed. For most antibiotics tested we found a signicant improvement in the validity of susceptibility testing. Only the testing on susceptible bacteria based on ampicillin and, to a minor degree, the testing on resistant bacteria based on mecillinam showed a reduction in the percentage of correct results after the intervention. For most antibiotics examined, the percentage of correct results after intervention was more than 95%. On closer examination it turned out that the ampicillin disc used in Sensicult was inactive. Quality control of a sample of discs from the same ampicillin batch was performed at the bacteriological laboratory, and revealed that the minimal inhibitory concentration (MIC) for E. coli was not obtained. The total percentages of correct Sensicult results before and after the intervention were 82% and 98%, respectively (Wilcoxon, p B 0.001). For Iso-Res agar the corresponding gures were 90% and 96%, respectively (Wilcoxon, p B 0.05). Table II gives the clinically relevant results of the intervention and the percentages of correct results and predictive values after the intervention in accordance with the two susceptibility methods performed (Iso-Res agar and Sensicult) and the type of bacteria tested (susceptible or resistant). With the exception of ampicillin testing based on Sensicult, the percentage of correct results was 90% or more. For Iso-Res agar the predictive value of testing a bacterial strain as susceptible ranged from 94% (ampicillin) to 100% (sulphamethizole and trimethoprim) and the predictive values of testing a bacterial strain as resistant ranged from 86% (nitrofurantoin) to 97% (ampicillin). For Sensicult, the predictive value of testing a bacterial strain as susceptible ranged from 98% (mecilinnam) to 100% (sulphamethizole, ampicillin and trimetroprim); as resistant it ranged from 62% (ampicillin) to 92% (trimetroprim). The low predictive Scand J Prim Health Care 2000; 18
Resistant bacteria Before intervention 100 (96.1–100) 91.5 (83.9–96.3) 97.2 (85.5–99.9) 80.0 (68.7–88.6) 94.4 (72.7–99.6) –
After intervention 100 (95.9–100) 96.1 (91.7–98.6) 95.6 (84.9–99.5) 90.5 (69.6–98.8) 100 (91.8–100) –
value of resistant bacteria for Sensicult (62%) was due to false results deriving from the inactive ampicillin disc, as mentioned above. If the results deriving from the ampicillin susceptibility were excluded from the analysis, there was no signicant difference in validity between the two tests.
DISCUSSION This study suggests that the validity of susceptibility testing in general practice may be improved by instructions and standardisation of the methods in use. We found a signicant improvement for both susceptibility methods. Prior to the study we agreed on an acceptable level at less than 5% false results, and this goal of quality was obtained for both systems tested. For Sensicult, however, this result was only obtained after exclusion of the results based on the ampicillin disc, deriving from a faulty batch. Problems with validity of susceptibility testing caused by inactivation of ampicillin discs have also been found in other studies of susceptibility testing based on Sensicult. Dornbusch thus showed that ampicillin discs are very sensitive to inappropriate transport and storing conditions, and proposed to store ampicillin discs at a temperature of ¼20°C (2). However, in this study we recommended that practices store discs in accordance with the instructions from the company (Biodisks at 4°C and Neo-Sensitabs at room temperature). Most studies of the validity of susceptibility testing in general practice have been performed without prior instruction or experience in the method. Few studies have evaluated the effect of a training programme for susceptibility testing and results are inconsistent. Thus, Dornbusch did not nd any effect of a training programme for Sensicult performed in general practice (2). On the other hand, Gunnarsson et al. (7) found that a few hours training resulted in a signicant reduction in the frequency of false Sensicult results. In our study we found a signicant improvement of the validity of Sensicult after inter-
86 (77–97) 62 (53–70) 85 (74–99) – 92 (73–99) – 100 (97–100) 100 (90–100) 98 (91–100) – 100 (97–100) – 100 (92–100) 100 (96–100) 96 (78–100) – 100 (85–100) – (91–99) (29–50) (85–98) (92–99) (95–100) (93–99) 96 39 94 97 99 97 (84–99) (88–99) (65–97) (65–97) (77–100) – Sensicult: Store uro-tubes vacuum packed at room temperature.
92 97 91 86 95 (97–100) (87–98) (94–100) (84–99) (97–100) – 100 94 99 95 100 (92–100) (81–97) (77–99) (70–98) (84–100) – 100 91 95 90 100 (93–99) (92–100) (94–100) (81–99) (96–100) (91–98) 97 98 98 93 100 96 Sulfamethizol Ampicillin Mecilinnam Nitrofurantoin Trimetroprim Ooxacin
Test indicating resistant bacteria Test indicating susceptible bacteria Resistant bacteria Susceptible bacteria Test indicating resistant bacteria Test indicating susceptible bacteria Resistant bacteria Susceptible bacteria
Predictive values (SE) Percentage of correct results (SE) Predictive value (SE) Percentage of correct results (SE)
Sensicult method IsoRes agar method
Table II. Results of Iso-Res agar and Sensicult susceptibility testing of urine specimens after intervention. The percentage of correct results (95% condence interval) and the predictive values of susceptibility testing are shown for each antibiotic. Values obtained from the bacteriological laboratory were used as reference.
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vention. About 98% of all bacteria were classied correctly after the training programme (when excluding the false ampicillin results), compared to 82% before training. The validity of Iso-Res agar was superior to Sensicult before the intervention was launched, but after the intervention there was no difference in the validity between Iso-Res agar and Sensicult. When performing susceptibility testing it is imperative that the method is standardised down to the last detail, and all specimens and remedies should be stored under the right conditions (temperature, humidity) before use (8). Otherwise the validity of the test may deteriorate. Furthermore, interpretation of a susceptibility test requires some knowledge of bacteriology and pharmacology. Experience from visits to GP surgeries and examination of susceptibility testing revealed some possible reasons for false results. An important source of false results was variation in the size of inoculum. If the inoculum is too big, the density of the colonies on the plate will increase, which in turn will lead to a decrease of the inhibition zones and vice versa for too small an inoculum. Preferably, the bacteria should be regularly spread on the plate giving rise to semi-conuent colonies (‘‘shoulder by shoulder’’). With the dip-slide technique the inoculum cannot be standardised as in the ordinary disc diffusion test (8). This may explain some of the discrepancy in the validity between IsoRes agar and Sensicult before intervention. In order to standardise the inoculum we proposed practices for using a sterile swap to transfer urine from specimen to the Iso-Res agar. We found that this method, when performed in the bacteriological laboratory, resulted in a suitable number of colonies on the agar plate (not published). When comparing the validity of Sensicult and Iso-Resagar before introduction of the intervention, we found that Iso-Resagar showed the highest validity. After intervention, however, there was no signicant difference in validity between the two tests. When using Iso-Resagar it is possible to investigate the resistance to 6 – 7 antibiotics in one test, while the maximum number examined using Sensicult is four. The choice of which test to use in practice may depend on the price and the availability of instruction. For both tests, however, continuous monitoring of the validity is crucial. Another reason for false results could be caused by susceptibility testing of a mixture of bacteria. When a mixture of bacteria is present in the urine specimen the results obtained from a direct susceptibility testing are completely unreliable (15). Combinations of sensitive species may give rise to reactions interpreted as resistance. Therefore antibiotic susceptibility testing should always be limited to pure cultures. HoffScand J Prim Health Care 2000; 18
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mann et al. (16) found that only 2% of results were correct if a mixture of bacteria was tested in general practices. The estimates of validity found in this study were all based on monocultures of bacteria and may therefore be too optimistic. One of the limitations of our study is the use of a before–after design. The optimal design would have been a randomised controlled study focusing on practices that had received intervention and practices that had not. Indeed, the improvement of quality shown in this study could be caused by factors other than the effect of the specic intervention. The procedure of analysing the quality of susceptibility testing can thus, in itself, lead to a change in the quality of the test. Another limitation concerns the generalisation of our results. In this study we analysed test quality in a sample of GP surgeries that volunteered to participate in the study. Twenty-three out of 25 agreed to participate. However, not all GPs routinely perform susceptibility testing, and those that do may be more interested in microbiology than average. Therefore, one should be careful when generalising the results of the intervention to all practices. This study emphasises the importance of training and experience when performing susceptibility testing in general practice. The reliability of the results depends on the experience of the person who performs the procedure. Training, standardisation of methods and particular interest in bacteriology are important factors for a reliable result of susceptibility testing in general practice. In some countries a laboratory quality programme has been implemented as routine in general practice. However, we are not aware of any programme specically focusing on the quality of microbiological testing. Based on our experience with the instruction procedure we nd that it is realistic and clinically relevant to incorporate microbiological quality assurance as a routine procedure in a laboratory quality programme for general practice. CONCLUSION The validity of susceptibility testing in general practice is acceptable when preceded by standardisation and instruction programmes. More than 95% of results are correct and the validity of the test is equal for susceptible and resistant bacteria. Further studies should be performed to document the durability of these ndings and the efforts needed to achieve continuous acceptable quality. ACKNOWLEDGEMENTS Thanks to the laboratory technicians at Odense University Hospital for distributing urine specimens to Scand J Prim Health Care 2000; 18
the general practitioners. Thanks to the general practitioners and the staff in practice for participating in the study. Thanks to the companies for delivering free susceptibility tests and thanks to the County of Funen for nancially supporting the study (Grant 0742). Conicts of Interest: None. REFERENCES 1. Gunnes M. Diagnostikk ved urinveisinfeksjoner i almenpraksis. II. Almenpraktikerens grunnlag for diagnostikk og behandling (Diagnosis of urinary tract infections in general practice. II. The general practitioner’s basis for diagnosis and treatment). Tidsskr Nor Laegeforen 1986;106:22 4– 5. 2. Dornbusch K, Lindeberg B, Nord CE, Thunell S. Bacteriuria diagnosis and antibiotic susceptibility testing in a group practice by dipslide techniques. Chemotherapy 1979;25:22 7 – 32. 3. Kjaerulff E. Susceptibility testing performed in general practice by urinary tract infections. Scand J Prim Health Care 1986;4:20 5– 8. 4. Kolmos HJ, Little P. Controversies in management: should general practitioners perform diagnostic tests on patients before prescribing antibiotics? BMJ 1999;318:79 9 – 802. 5. Dornbusch K, Nord CE, Olsson B, Thunell S. Antibacterial susceptibility testing by the dip-slide technique: a methodological evaluation. Chemotherapy 1976;22:19 0 –202. 6. Bergan T, Bruun JN, Digranes A, Lingaas E, Melby KK, Sander J. Susceptibility testing of bacteria and fungi. Report from the ‘‘Norwegian Working Group on Antibiotics’’. Scand J Infect Dis Suppl 1997;103: 1– 36. 7. Gunnarsson C, Henning C, Naslund B, Wehlin M. Diagnostik och resistensprovning med hja¨lp av ‘‘dip-slides’’ vid urinva¨gsinfektioner (Diagnosis and resistance testing with dip slides in urinary infection). La¨kartidningen 1975;72:498 9– 91. 8. Ericsson HM, Sherris JC. Antibiotic sensitivity testing. Report of an international collaborative study. Acta Pathol Microbiol Scand [B]. Microbiol Immunol 1971 217:Suppl. 9. Johnson JR, Tiu FS, Stamm WE. Direct antimicrobial susceptibility testing for acute urinary tract infections in women. J Clin Microbiol 1995;33:231 6– 23. 10. Kallenius G, Dornbusch K, Hallander HO, Jakobsson K. Comparison of direct and standardized antibiotic susceptibility testing in bacteriuria. Chemotherapy 1981;27:9 9– 105. 11. Bjerrum L, Grinsted P, Søgaard Per, Petersen PH. Validity of susceptibility testing of uropathogenic bacteria in general practice. Br J Gen Pract 1999;49:82 1 – 2. 12. Ferry S, Burman LG, Holm SE. Uricult and Sensicult dipslides for diagnosis of bacteriuria and prediction of drug resistance in primary health care. Scand J Prim Health Care 1989;7:12 3 –8. 13. Degre M, Hovig B, Midtvedt T. Kan transportagarmetoden utnyttes til resistensbestemmelse ved urinveisinfeksjoner (Can the transport agar method be used for determination of resistance in urinary tract infections?). Tidsskr Nor Laegeforen 1977;97:24 8 –50. 14. Oakes AR, Badger R, Grove DI. Comparison of direct and standardized testing of infected urine for antimicrobial susceptibilities by disk diffusion. J Clin Microbiol 1994;32:4 0 –5. 15. Shahidi A, Ellner PD. Effect of mixed cultures on antibiotic susceptibility testing. Appl Microbiol 1969;18:76 6 – 70. 16. Hoffmann S, Mabeck CE, Vejlsgaard R, Mortensen I. Følsomhedsbestemmelse ved bakteriuri i almen praksis med Sensicult (Sensitivity testing in bacteriuria in general practice with Sensicult). Ugeskr Laeger 1982;144:9 7 – 100.
