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Should resistance to azole antifungals in vitro be interpreted as predicting clinical non-response?
Should resistance to azole antifungals in vitro be interpreted as predicting clinical non-response? Frank C Odds
Departmentof Bacteriologyand Mycology,JanssenResearchFoundation, Beerse,Belgium Abstract Published data relating clinical treatment outcomes to susceptibility of Candida species in vitro for the triazole antifungal agents fluconazole and itraconazole show a clear association between rates of treatment failure and rising minimal inhibitory concentrations for the infecting fungal isolate. However, more than 50% of patients infected with an isolate 'resistant' to a triazole by NCCLS breakpoint criteria respond successfully to treatment with the triazole. Data for antibacterial agents similarly show that the association between resistance in vitro and treatment failure in vivo is far less than perfect. Susceptibility testing therefore falls into a category similar to chat of weather forecasting. Despite good test standardization and sophisticated technology, the forecasts successfully predict trends, but cannot accurately foresee temperatures or levels of precipitation at a specific time in a specific location. Considerable progress has b e e n made in the standardization of antiffmgal susceptibility testing over t h e last 12 years. The main driving force for standardization has b e e n the Antifungal Susceptibility Testing Subcommittee of the U.S. National Committee for Clinical Laboratory Standards (NCCLS). Based on extensive experimental collaborations b e t w e e n specialist medical mycology laboratories, the NCCLS Subcommittee has defined a reference broth dilution method, M27-A, for determining minimal inhibitory concentrations (MICs) of five antifungal agents against clinically important yeasts. ~The M27-A reference standard d o c u m e n t also provides guidelines for interpretive MIC breakpoints to indicate reduced susceptibility and resistance to two widely used antifungal agents, itraconazolc and fluconazole. For these agents, at least, clinical microbiology laboratories are n o w in a position to undertake routine susceptibility testing in the context of patient management. This is, therefore, an appropriate time to examine the clinical predictive w o r t h of antifmlgal susceptibility testing. THE RATIONALE OF RESISTANCE TESTING Antimicrobial susceptibility testing, particularly testing done with antibacterial agents, has attained the status of a s i n e q u a n o n for therapeutic choices in infectious diseases. It is by no means u n c o m m o n to find practitioners w h o are unwilling to treat a patient with a particular agent unt~ they have seen laboratory result s proving that the patient's i ~ ing microbe is susceptible to the agent in vitro.The rationale
for such an attitude is easy to understand. The physician wants to select the agent offering the greatest chance of treatment success. There are many reasons w h y a particular antimicrobial agent may fail to cure an infection in the clinic. Broadly summarized, these include failure to achieve adequate concentrations of drug at the site of infection (for one of many reasons); reduced or hllpaired host defense mechanisms necessary to work in concert with the antimicrobial agent; and resistance of the infecting organism to the agent. Of these various reasons for treatment failure, only the last can be easily quantified by laboratory tests. A 'susceptibility' test is, therefore, really a test for resistance, and a finding of resistance to an agent in vitro is c o m m o n l y interpreted as an absolute contra-indication for treatment with that agent. In truth, the extrapolation of a finding of resistance in vitro to clinical treatment decisions is complicated. Resistance to an agent is a quantitative, not a qualitative p h e n o m e n o n , and the customary simplification of MIC data into groups of susceptibility based on interpretive breakpoints still reflects the quantitative nature of the result by defining an intermediate (I) region of susceptibility that lies b e t w e e n what is regarded as unequivocal sensitivity to an agent (S) and unequivocal resistance (R). The true definition of resistance in vitro also needs to take account of predictions of drug levels: if a blood level greater than a measured minimal inhibitory concentration (MIC) can be achieved, then an organism should be, in theory, susceptible to that level in vivo. Moore et aP have shown very elegantly that, at least for aminoglycoside antibacterials, the ratio of peak seruna concentration to MIC is a m u c h better predictor of clinical o u t c o m e than MIC alone. While the doctrine of 'resistance in vitro equals clinical failure' is encountered very widely as a clinical guideline, by no means all investigators have accepted it unquestioningly. Indeed, some experts in the susceptibility testing field have occasionally p r o n o u n c e d cautions. '... one frequently sees that in daily practice MICs are poorly lmderstood and wrongly interpreted'? 'The problem of extrapolating laboratory results to the clinical situation presents such a minefield of difficulties that microbiologists usually prefer to concentrate on obtaining reproducible estimates of antimicrobial susceptibility in the laboratory ... and to leave the problem of clinical relevance to the physician'. 4 CORRELATIONS BETWEEN SUSCEPTIBILITY IN VITRO AND CLLNICAL TREATMENT OUTCOMES The question of correlations b e t w e e n susceptibility test results in vitro and the o u t c o m e of treatment in the clinic has b e e n addressed for two triazole antiflmgal agents, fluconazole and itraconazole, in a n u m b e r of recent publications.All but a few of these studies deal w i t h yeast isolates from HIV-positive patients w i t h oropharyngeal C a n d i d a infections treated with fluconazole. Most of t h e m s h o w good to excellent statistical correlations b e t w e e n treatment results and MIC values, with higher rates of treatment failure associated with higher MICs. Table 1 summarizes publications of this type in w h i c h the susceptibility testing was done by the NCCLS reference m e t h o d or a minor variant of
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this m e t h o d . (Some studies w e r e e x c l u d e d from t h e table, e v e n t h o u g h t h e NCCLS m e t h o d was used, b e c a u s e t h e range of azole c o n c e n t r a t i o n s t e s t e d did n o t allow for calculations b a s e d o n t h e NCCLS-recommended breakpoints, or t h e data p r e s e n t e d did n o t allow for e x t r a c t i o n of results in t h e f o r m a t of Table 1.) It is self-evident from t h e data in Table 1 that, w i t h few exceptions, t h e p e r c e n t a g e s of t r e a t m e n t s w i t h successful o u t c o m e s are highest a m o n g p a t i e n t s infected w i t h isolates in t h e 'S' category, a n d lowest a m o n g p a t i e n t s infected w i t h isolates in t h e 'R' category. It is also serf-evident t h a t b y n o m e a n s 100% o f p a t i e n t s infected w i t h a 'resistant' yeast fail to r e s p o n d to antifungal t r e a t m e n t w i t h w h a t w o u l d normally b e r e g a r d e d as a n ' i n a p p r o p r i a t e ' agent. Of t h e total of 55 p a t i e n t s (Table 1) infected w i t h isolates at above t h e itraconazole resistance breakpoint, 56% w e r e scored as treatm e n t successes. The equivalent figure for fluconazole was 41% of 92 patients infected w i t h resistant isolates (data from ref. 7 o m i t t e d to avoid duplication). O f course, t h e data in Table 1 r e p r e s e n t several oversimplications: n o a c c o u n t is t a k e n of differences in doses given for t r e a t m e n t b e t w e e n studies or MIC categories; n o a c c o u n t is t a k e n of different criteria used to define 'success' a n d 'failure'; a n d data have b e e n ' s h o e - h o r n e d ' into MIC categories according to NCCLS breakpoints, regardless of different b r e a k p o i n t s p r o p o s e d b y t h e authors of t h e studies tabulated. Moreover, different p a t i e n t types are s o m e t i m e s b e i n g compared. However, t h e reality of m u c h routine practice is s u c h that m a n y fungal isolates (including fungi o t h e r t h a n C a n d i d a species) will b e t e s t e d for susceptibility to fluconazole a n d itraconazole b y m e t h o d M27-A, some will b e f o u n d to b e resistant according to NCCLS b r e a k p o i n t criteria, a n d t r e a t m e n t will n o t h e instituted o n t h e basis of that finding, regardless of o t h e r relevant factors. T h e largest g r o u p s of data s u m m a r i z e d in Table 1 are t h o s e p u b l i s h e d b y t h e NCCLS s u b c o m m i t t e e in t h e i r carefully r e a s o n e d a c c o u n t of b r e a k p o i n t d e t e r m i n a t i o n s ? ° With data supplied b y t h e m a n u f a c t u r e r s of t h e t w o drugs from t h e i r clinical trials (involving p r e d o m i n a n t l y o r o p h a r y n g e a l
C a n d i d a infections in HIV-positive patients), t h e subc o m m i t t e e calculated t h e p e r c e n t a g e of positive clinical r e s p o n s e s at each level of MIC. T h e t r e n d of t h e results was clear: as t h e MIC for t h e infecting C a n d i d a strain rose, t h e rate of clinical success declined. Resistance b r e a k p o i n t s w e r e designated as t h o s e MIC values at w h i c h t r e a t m e n t success rates first s h o w e d a s h a r p fall. In this way,'resistance' in vitro correlates w i t h a n e n h a n c e d probability of t r e a t m e n t failure, a result that fltrther s u p p o r t s t h e clinical value of t h e test. However, for any individual p a t i e n t infected w i t h a 'resistant' yeast isolate, t h e probability of t r e a t m e n t failure is n o t great: for b o t h itraconazole a n d fluconazole, as in several of t h e smaller studies tabulated, just over hal/" of t h e p a t i e n t s infected w i t h fungi resistant to t h o s e agents w e r e s c o r e d as clinical t r e a t m e n t successes. This p a r a d o x is b y n o m e a n s new, or exclusive to t h e antifungal field. It has b e e n d o c u m e n t e d repeatedly for antibacterial agents. J o h n s o n 11lists a n u m b e r of publications that correlate t r e a t m e n t o u t c o m e s w i t h antibacterial susceptibility testing. Table 2 stmunarizes this selection of results relating susceptibility to antibacterial agents in vitro w i t h results of t r e a t m e n t in t h e clinic. Table 3 summarizes t w o f u r t h e r studies in w h i c h t r e a t m e n t o u t c o m e s w e r e related to t h e ' a p p r o p r i a t e n e s s ' or o t h e r w i s e o f antimicrobial t h e r a p y u s e d in t h e clinic: susceptibility test results w e r e a m a j o r c o m p o n e n t of t h e assessment of appropriateness. T h e doctrine of 'resistance in vitro equals clinical failure' is elegantly u p h e l d inTable 2 w i t h results s u c h as t h o s e of Lorian et al,12 b u t t h e o t h e r data in Tables 2 a n d 3 m o r e closely r e s e m b l e t h o s e for fluconazole and itraeonazole s h o w n in Table 1. T h e r e is almost always a decrease in clinical success associated w i t h r e d u c e d susceptibility in vitro or i n a p p r o p r i a t e antimicrobial choices, b u t no-one could i n t e r p r e t t h e results as d e m o n s t r a t i n g that resistance in vitro reliably predicts a poor treatment outcome. T h e studies p r e s e n t e d in Tables 1-3 are n o t a n exhaustive list o f t h e i r type, b u t t h e y s h o w t h a t t h e t h e o r e t i c a l basis for a s s u m i n g a clinically p r e d i c t i v e value of antimicrobial resistance in vitro is b y n o m e a n s u n i f o r m l y s o u n d . T h e
Table 2 Published examples of correlations between antibacterial MlCs and clinical outcome of antibacterial t r e a t m e n t
Miscellaneous antibacterials MIC category
Improvement (95% CIt) n--
S
Antibacterials*
271
Survival (95% CIt) n=
81% (75-85)
Cefoperazone
88
Eradication (95% CIt) n=
25% (I 6-35)
Carbenicillin
58
Eradication (95% CI~) n=
72% (59-83)
20
Clinical setting Reference
27
4% (0-19)
9
67% (30-93)
28
50% (31-69)
80
Eradication (95% CIt) n=
90% (68-99)
I R
Cefotaxime
29% (I 9-40)
1282
89% (87-9 I)
II 5
78% (68--85)
43
65% (49-79)
Misc. bacterial infections
Pseudomonas
bacteremia
Urinary tract infections
Urinary tract infections
Misc. bacterial infections
12
13
14
14
15
*Gentamicin, carbenicillin or colistin. t95% confidence intervals determined by binomial approximation.
© 1998 Harcourt Brace & Co.Ltd DrugResistance Updates (1998) I, 1I - / 5
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Table 3 Some published correlations between appropriateness* of antimicrobial therapy and clinical outcome: two studies with miscellaneous agents
% Survival
% Survival
(95% Cl*)
(9S% Ci*)
cases, t h e organism d e t e r m i n e d to b e resistant may n o t b e t h e organism responsible for t h e infection. In diseases s u c h as o r o p h a r y n g e a l Candida infections, species s u c h as C.glabrata a n d C. krusei are often isolated from oral samples b u t t h e r e is d o u b t w h e t h e r t h e y always c o n t r i b u t e to s y m p t o m a t i c pathology. 22.23
Therapy deemed
n=
n=
Appropriate
431
81% (76-84)
725
64% (61-68)
CONCLUSIONS
inappropriate
172
63% (55-70)
167
56% (48-63)
179
61% (53-68)
T h e very t e r m ' b r e a k p o i n t ' c o n t r i b u t e s to t h e traditional image of antimicrobial susceptibility testing as a sharp-edged razor, capable of differentiating a n a p p r o p r i a t e from an i n a p p r o p r i a t e treatment. T h e reality, as e v i d e n c e d b y m a n y publications t h a t actively analyze t h e association b e t w e e n susceptibility in vitro a n d clinical t r e a t m e n t results, is t h a t susceptibility testing is p e r h a p s analogous to w e a t h e r forecasts. T h e m e t h o d s u s e d in w e a t h e r forecasting, as w i t h susceptibility testing, are o f t e n b a s e d o n sophisticated technology.They p r o v i d e a n excellent statistical basis for general w a r n i n g s of adverse events b u t t h e y c a n n o t accurately predict t e m p e r a t u r e or precipitation in any specific location at any specific time. In clinical practice it is i m p o r t a n t for physicians to a c k n o w l e d g e that t h e r a p e u t i c success w i t h fungal diseases is partly or largely a f u n c t i o n of t h e i m m u n e status of t h e patient, t h e nature of t h e underlying disease, a n d o t h e r factors u n r e l a t e d to susceptibility of t h e infecting a g e n t in vitro. 1°'2~25A laboratory finding of resistance should b e a w a r n i n g for t h e clinician, b u t n o t an i m m e d i a t e contraindication for a particular agent.
None
Clinical setting
Reference
bacteremia
Gram-negative bacteremia
16
17
*"Appropriate" therapy means use of an agent for which the bacterial isolate was susceptible in vitro, or for which the isolated species is usually found susceptible and which could be given to achieve sufficient blood or tissue concentrations in the given case of infection. *95% confidence intervals determined by binomial approximation.
association b e t w e e n microbial resistance a n d t r e a t m e n t failure is b e t t e r for s o m e t y p e s of i n f e c t i o n t h a n for others, a n d it is b e t t e r for s o m e m i c r o b i a l species t h a n for others. T h o r n s b e r r y et a115 b r o k e d o w n t h e i r large d a t a b a s e for c e f o t a x i m e MICs versus t r e a t m e n t o u t c o m e b y different bacterial g r o u p s ( t h e data in Table 2 s h o w t h e overall results). For Gram-negative bacilli o t h e r t h a n Enterob a c t e r i a c e a e a n d Pseudomonas spp., for example, t h e y f o u n d a h i g h c o r r e l a t i o n b e t w e e n susceptibility in vitro a n d good t r e a t m e n t o u t c o m e s , w i t h e r a d i c a t i o n of t h e b a c t e r i a in 98% of p a t i e n t s i n f e c t e d w i t h s u s c e p t i b l e isolates. However, e v e n for t h e s e organisms, 50% e r a d i c a t i o n was d o c u m e n t e d for t h e p a t i e n t s i n f e c t e d w i t h isolates resistant to c e f o t a x i m e in vitro. For t h e e n t e r o c o c c i as a group, t h e clinical e r a d i c a t i o n rate for 'resistant' isolates w a s actually h i g h e r t h a n for ' s u s c e p t i b l e ' isolates (82% versus 65%), b u t this result reflects a relatively l o w n u m b e r of resistant isolates, as is also s e e n in Table 2 for Pseudomonas spp. in .the data f r o m Flick a n d Cluff. 13 In t h e i r large study of c a n d i d e m i a , Rex et al 7 f o u n d an. inverse c o r r e l a t i o n b e t w e e n antifungal susceptibility test results a n d clinical t r e a t m e n t success (this study i n v o l v e d a m p h o t e r i c i n B as well as fluconazole). In t h e same way as reasons c a n b e postulated for treatm e n t failures w i t h susceptible organisms, t r e a t m e n t successes w i t h resistant ones m i g h t b e e x p l a i n e d in several ways. Subinhibitory effects of amimicrobial agents m i g h t tip t h e balance in favor of t h e host. For b o t h antibacterial agents 18,19 a n d a n antifungal agent, 2° s u b i n h i b i t o r y c o n c e n t r a t i o n s of inhibitors have b e e n s h o w n to alter microbial e x p r e s s i o n of toxins or surface molectfles that may b e involved in t h e infective process. An agent may interact synergistically w i t h a host m o l e c u l e a n d thus eradicate an o t h e r w i s e resistant organism: s u c h effects have b e e n d e s c r i b e d at least for g e n t a m i c i n a n d h u m a n amniotic fluid. ~1 Finally, in some
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References I. National Committee for Clinical Laboratory Standards. Reference method for broth dilution antifungal susceptibility testing of yeasts.Approved standard M27-A. NCCL5 1997. Villanova, PA. 2. Moore RD, Lietman PS, Smith CR. Clinical response to aminoglycoside therapy: importance of the ratio of peak concentration to minimal inhibitory concentration. J Infect Dis 1987; 155:93-99. 3. Amsterdam D. Susceptibility testing of antimicrobials in liquid media. In: LorianV (ed).Antibiotics in Laboratory Medicine, 4th edn. Baltimore: William & Wilkins, 1996: 52- I I I. 4. Greenwood D. In vitro veritas. Antimicrobial susceptibility tests and their clinical relevance.J Infect Dis 1981; 144: 380-385. 5. Barchiesi F,Hollis RJ, Mcgough DA, Scalise G, Rinaldi MG, Pfaller MA. DNA subtypes and fluconazole susceptibilities of Candida albicans isolates from the oral cavities of patients with AIDS. Clin Infect Dis 1995; 20: 634-640. 6. Espinel-lngroffA, Rodriguez-Tudela JL, Martinez-Suarez JV. Comparison of two alternative microdilution procedures with the National Committee for Clinical Laboratory Standards reference macrodilution method M27-P for in vitro testing of fluconazole-resistant and -susceptible isolates of Candida albicans. J Clin Microbiol 1995; 33:3154-3158.
7. Rex JH, Pfaller MA, BarryAL, Nelson PW, Webb CD. Antifungal susceptibility testing of isolates from a randomized, multicenter trial of fluconazole versus amphotericin B as treatment of nonneutropenic patients with candidemia.Antimicrob Agents Chemother 1995; 39: 40-44. 8. Phillips R Zemcov J, Mahmood W, Montaner JSG, Craib K, Clarke AM. Itraconazole cyclodextrin solution for tquconazole refractory oropharyngeal candidiasis in AIDS: correlation of clinical response with in vitro susceptibility.AIDS 1996; I 0:1369-1376. 9. Quereda C, Polanco AM, Giner C et al. Correlation between in vitro resistance to fluconazole and clinical outcome of oropharyngeal candidiasis in HIV-infected patients. Eur J Clin Microbiol Infect Dis 1996; 15: 30-37. 10. Rex JH, Pfaller MA, Galgiani JN et al. Development of interpretive breakpoints for antifungal susceptibility testing: conceptual framework and analysis of in vitro-in vivo correlation data for fluconazole, itraconazole and Candida infections. Clin Infect Dis 1997; 24: 235-247. I I. Johnson CC. In vitro testing: correlations of bacterial susceptibility, body fluid levels, and effectiveness of antibacterial therapy. In: Lorian V (ed).Antibiotics in Laboratory Medicine, 4th edn. Baltimore: William & Wilkins, 1996:813-834. 12. Lorian V, Burns L, Ernst J. Predictive value of susceptibility tests for the outcome of antibacterial therapy.J Antimicrob Chemother 1990; 25:175-18 I. 13. Flick MR, Cluff LE. Pseudomonas bacteremia: review of 108 cases. Am J Med 1976; 60:501-508. 14. GerberAU, CraigWA.Worldwide clinical experience with cefoperazone. Drugs 198 I; 22 (Suppl I): 108-I 18. 15. Thornsberry C,Jones RN, BarryAL, Fuchs PC.Antimicrobial susceptibility tests with cefotaxime and correlation with clinical bacteriologic response. Rev Infect Dis 1982; 4 (Suppl): $316-$324.
16. Kreger BE, Craven DE, McCabe WR. Gram-negative bacteremia. IV. Re-evaluation of clinical features and treatment in 612 patients. Am J Med 1980; 68: 344-354. 17. Bryan CS, Reynolds KL, Brenner ER.Analysis of I 186 episodes of gram-negative bacteremia in non-university hospitals: the effects of antimicrobial therapy. Rev Infect Dis 1983; 5: 629-638. 18. Dupont MJ, Lapointe JR. Effects of Pseudomonas aeruginosa alginate expression of direct plating and culture of fresh cystic fibrosis sputum on to Pseudomonas isolation agar containing subinhibitory concentrations of roxithromycin and rifampicin.J Antimicrob Chemother 1995; 36:231-236. 19. HostackaA. Postantibiotic effects of subinhibitory concentrations of some antibiotics and their influence on Pseudomonas aeruginosa enzymic activity. Folia Microbiol 1996; 4 I: 39-42. 20. Angiolella L, Facchin M, Stringaro A, Maras B, Simonetti N, Cassone A. Identification of a glucan-associated enolase as a main cell wall protein of Candida albicans and an indirect target of lipopeptide antimycotics.J Infect Dis 1996; 173: 684-690. 2 I. Miglioli PA, Schoffel U, Gianfranceschi L. The in vitro synergistic inhibitory effect of human amniotic fluid and gentamicin on growth of Escherichia coli. Chemotherapy 1996; 42: 206-209. 22. Maenza JR, MerzWG, Romagnoli MJ, Keruly JC, Moore RC, Gallant JE. Infection due to fluconazole-resistant Candida in patients with AIDS: prevalence and microbiology. Clin Infect Dis 1997; 24: 28-34. 23. Dronda F,Alonsosanz M, Laguna F et al. Mixed oropharyngeal candidiasis due to Candida albicans and non-albicans Candida strains in HIV-infected patients.J Clin Microbiol Infect Dis 1996; 15:446-452. 24. Pittrow L, PenkA. Plasma and tissue concentrations of fluconazole and their correlation to breakpoints. Mycoses 1997;40:25-32. 25. Ghannoum MA, RexJH, GalgianiJN. Susceptibility testing of fungi: current status of correlation of in vitro data with clinical outcome. J Clin Microbiol 1996; 34: 489-495.
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Departmentof Bacteriologyand Mycology,JanssenResearchFoundation, Beerse,Belgium Abstract Published data relating clinical treatment outcomes to susceptibility of Candida species in vitro for the triazole antifungal agents fluconazole and itraconazole show a clear association between rates of treatment failure and rising minimal inhibitory concentrations for the infecting fungal isolate. However, more than 50% of patients infected with an isolate 'resistant' to a triazole by NCCLS breakpoint criteria respond successfully to treatment with the triazole. Data for antibacterial agents similarly show that the association between resistance in vitro and treatment failure in vivo is far less than perfect. Susceptibility testing therefore falls into a category similar to chat of weather forecasting. Despite good test standardization and sophisticated technology, the forecasts successfully predict trends, but cannot accurately foresee temperatures or levels of precipitation at a specific time in a specific location. Considerable progress has b e e n made in the standardization of antiffmgal susceptibility testing over t h e last 12 years. The main driving force for standardization has b e e n the Antifungal Susceptibility Testing Subcommittee of the U.S. National Committee for Clinical Laboratory Standards (NCCLS). Based on extensive experimental collaborations b e t w e e n specialist medical mycology laboratories, the NCCLS Subcommittee has defined a reference broth dilution method, M27-A, for determining minimal inhibitory concentrations (MICs) of five antifungal agents against clinically important yeasts. ~The M27-A reference standard d o c u m e n t also provides guidelines for interpretive MIC breakpoints to indicate reduced susceptibility and resistance to two widely used antifungal agents, itraconazolc and fluconazole. For these agents, at least, clinical microbiology laboratories are n o w in a position to undertake routine susceptibility testing in the context of patient management. This is, therefore, an appropriate time to examine the clinical predictive w o r t h of antifmlgal susceptibility testing. THE RATIONALE OF RESISTANCE TESTING Antimicrobial susceptibility testing, particularly testing done with antibacterial agents, has attained the status of a s i n e q u a n o n for therapeutic choices in infectious diseases. It is by no means u n c o m m o n to find practitioners w h o are unwilling to treat a patient with a particular agent unt~ they have seen laboratory result s proving that the patient's i ~ ing microbe is susceptible to the agent in vitro.The rationale
for such an attitude is easy to understand. The physician wants to select the agent offering the greatest chance of treatment success. There are many reasons w h y a particular antimicrobial agent may fail to cure an infection in the clinic. Broadly summarized, these include failure to achieve adequate concentrations of drug at the site of infection (for one of many reasons); reduced or hllpaired host defense mechanisms necessary to work in concert with the antimicrobial agent; and resistance of the infecting organism to the agent. Of these various reasons for treatment failure, only the last can be easily quantified by laboratory tests. A 'susceptibility' test is, therefore, really a test for resistance, and a finding of resistance to an agent in vitro is c o m m o n l y interpreted as an absolute contra-indication for treatment with that agent. In truth, the extrapolation of a finding of resistance in vitro to clinical treatment decisions is complicated. Resistance to an agent is a quantitative, not a qualitative p h e n o m e n o n , and the customary simplification of MIC data into groups of susceptibility based on interpretive breakpoints still reflects the quantitative nature of the result by defining an intermediate (I) region of susceptibility that lies b e t w e e n what is regarded as unequivocal sensitivity to an agent (S) and unequivocal resistance (R). The true definition of resistance in vitro also needs to take account of predictions of drug levels: if a blood level greater than a measured minimal inhibitory concentration (MIC) can be achieved, then an organism should be, in theory, susceptible to that level in vivo. Moore et aP have shown very elegantly that, at least for aminoglycoside antibacterials, the ratio of peak seruna concentration to MIC is a m u c h better predictor of clinical o u t c o m e than MIC alone. While the doctrine of 'resistance in vitro equals clinical failure' is encountered very widely as a clinical guideline, by no means all investigators have accepted it unquestioningly. Indeed, some experts in the susceptibility testing field have occasionally p r o n o u n c e d cautions. '... one frequently sees that in daily practice MICs are poorly lmderstood and wrongly interpreted'? 'The problem of extrapolating laboratory results to the clinical situation presents such a minefield of difficulties that microbiologists usually prefer to concentrate on obtaining reproducible estimates of antimicrobial susceptibility in the laboratory ... and to leave the problem of clinical relevance to the physician'. 4 CORRELATIONS BETWEEN SUSCEPTIBILITY IN VITRO AND CLLNICAL TREATMENT OUTCOMES The question of correlations b e t w e e n susceptibility test results in vitro and the o u t c o m e of treatment in the clinic has b e e n addressed for two triazole antiflmgal agents, fluconazole and itraconazole, in a n u m b e r of recent publications.All but a few of these studies deal w i t h yeast isolates from HIV-positive patients w i t h oropharyngeal C a n d i d a infections treated with fluconazole. Most of t h e m s h o w good to excellent statistical correlations b e t w e e n treatment results and MIC values, with higher rates of treatment failure associated with higher MICs. Table 1 summarizes publications of this type in w h i c h the susceptibility testing was done by the NCCLS reference m e t h o d or a minor variant of
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this m e t h o d . (Some studies w e r e e x c l u d e d from t h e table, e v e n t h o u g h t h e NCCLS m e t h o d was used, b e c a u s e t h e range of azole c o n c e n t r a t i o n s t e s t e d did n o t allow for calculations b a s e d o n t h e NCCLS-recommended breakpoints, or t h e data p r e s e n t e d did n o t allow for e x t r a c t i o n of results in t h e f o r m a t of Table 1.) It is self-evident from t h e data in Table 1 that, w i t h few exceptions, t h e p e r c e n t a g e s of t r e a t m e n t s w i t h successful o u t c o m e s are highest a m o n g p a t i e n t s infected w i t h isolates in t h e 'S' category, a n d lowest a m o n g p a t i e n t s infected w i t h isolates in t h e 'R' category. It is also serf-evident t h a t b y n o m e a n s 100% o f p a t i e n t s infected w i t h a 'resistant' yeast fail to r e s p o n d to antifungal t r e a t m e n t w i t h w h a t w o u l d normally b e r e g a r d e d as a n ' i n a p p r o p r i a t e ' agent. Of t h e total of 55 p a t i e n t s (Table 1) infected w i t h isolates at above t h e itraconazole resistance breakpoint, 56% w e r e scored as treatm e n t successes. The equivalent figure for fluconazole was 41% of 92 patients infected w i t h resistant isolates (data from ref. 7 o m i t t e d to avoid duplication). O f course, t h e data in Table 1 r e p r e s e n t several oversimplications: n o a c c o u n t is t a k e n of differences in doses given for t r e a t m e n t b e t w e e n studies or MIC categories; n o a c c o u n t is t a k e n of different criteria used to define 'success' a n d 'failure'; a n d data have b e e n ' s h o e - h o r n e d ' into MIC categories according to NCCLS breakpoints, regardless of different b r e a k p o i n t s p r o p o s e d b y t h e authors of t h e studies tabulated. Moreover, different p a t i e n t types are s o m e t i m e s b e i n g compared. However, t h e reality of m u c h routine practice is s u c h that m a n y fungal isolates (including fungi o t h e r t h a n C a n d i d a species) will b e t e s t e d for susceptibility to fluconazole a n d itraconazole b y m e t h o d M27-A, some will b e f o u n d to b e resistant according to NCCLS b r e a k p o i n t criteria, a n d t r e a t m e n t will n o t h e instituted o n t h e basis of that finding, regardless of o t h e r relevant factors. T h e largest g r o u p s of data s u m m a r i z e d in Table 1 are t h o s e p u b l i s h e d b y t h e NCCLS s u b c o m m i t t e e in t h e i r carefully r e a s o n e d a c c o u n t of b r e a k p o i n t d e t e r m i n a t i o n s ? ° With data supplied b y t h e m a n u f a c t u r e r s of t h e t w o drugs from t h e i r clinical trials (involving p r e d o m i n a n t l y o r o p h a r y n g e a l
C a n d i d a infections in HIV-positive patients), t h e subc o m m i t t e e calculated t h e p e r c e n t a g e of positive clinical r e s p o n s e s at each level of MIC. T h e t r e n d of t h e results was clear: as t h e MIC for t h e infecting C a n d i d a strain rose, t h e rate of clinical success declined. Resistance b r e a k p o i n t s w e r e designated as t h o s e MIC values at w h i c h t r e a t m e n t success rates first s h o w e d a s h a r p fall. In this way,'resistance' in vitro correlates w i t h a n e n h a n c e d probability of t r e a t m e n t failure, a result that fltrther s u p p o r t s t h e clinical value of t h e test. However, for any individual p a t i e n t infected w i t h a 'resistant' yeast isolate, t h e probability of t r e a t m e n t failure is n o t great: for b o t h itraconazole a n d fluconazole, as in several of t h e smaller studies tabulated, just over hal/" of t h e p a t i e n t s infected w i t h fungi resistant to t h o s e agents w e r e s c o r e d as clinical t r e a t m e n t successes. This p a r a d o x is b y n o m e a n s new, or exclusive to t h e antifungal field. It has b e e n d o c u m e n t e d repeatedly for antibacterial agents. J o h n s o n 11lists a n u m b e r of publications that correlate t r e a t m e n t o u t c o m e s w i t h antibacterial susceptibility testing. Table 2 stmunarizes this selection of results relating susceptibility to antibacterial agents in vitro w i t h results of t r e a t m e n t in t h e clinic. Table 3 summarizes t w o f u r t h e r studies in w h i c h t r e a t m e n t o u t c o m e s w e r e related to t h e ' a p p r o p r i a t e n e s s ' or o t h e r w i s e o f antimicrobial t h e r a p y u s e d in t h e clinic: susceptibility test results w e r e a m a j o r c o m p o n e n t of t h e assessment of appropriateness. T h e doctrine of 'resistance in vitro equals clinical failure' is elegantly u p h e l d inTable 2 w i t h results s u c h as t h o s e of Lorian et al,12 b u t t h e o t h e r data in Tables 2 a n d 3 m o r e closely r e s e m b l e t h o s e for fluconazole and itraeonazole s h o w n in Table 1. T h e r e is almost always a decrease in clinical success associated w i t h r e d u c e d susceptibility in vitro or i n a p p r o p r i a t e antimicrobial choices, b u t no-one could i n t e r p r e t t h e results as d e m o n s t r a t i n g that resistance in vitro reliably predicts a poor treatment outcome. T h e studies p r e s e n t e d in Tables 1-3 are n o t a n exhaustive list o f t h e i r type, b u t t h e y s h o w t h a t t h e t h e o r e t i c a l basis for a s s u m i n g a clinically p r e d i c t i v e value of antimicrobial resistance in vitro is b y n o m e a n s u n i f o r m l y s o u n d . T h e
Table 2 Published examples of correlations between antibacterial MlCs and clinical outcome of antibacterial t r e a t m e n t
Miscellaneous antibacterials MIC category
Improvement (95% CIt) n--
S
Antibacterials*
271
Survival (95% CIt) n=
81% (75-85)
Cefoperazone
88
Eradication (95% CIt) n=
25% (I 6-35)
Carbenicillin
58
Eradication (95% CI~) n=
72% (59-83)
20
Clinical setting Reference
27
4% (0-19)
9
67% (30-93)
28
50% (31-69)
80
Eradication (95% CIt) n=
90% (68-99)
I R
Cefotaxime
29% (I 9-40)
1282
89% (87-9 I)
II 5
78% (68--85)
43
65% (49-79)
Misc. bacterial infections
Pseudomonas
bacteremia
Urinary tract infections
Urinary tract infections
Misc. bacterial infections
12
13
14
14
15
*Gentamicin, carbenicillin or colistin. t95% confidence intervals determined by binomial approximation.
© 1998 Harcourt Brace & Co.Ltd DrugResistance Updates (1998) I, 1I - / 5
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Table 3 Some published correlations between appropriateness* of antimicrobial therapy and clinical outcome: two studies with miscellaneous agents
% Survival
% Survival
(95% Cl*)
(9S% Ci*)
cases, t h e organism d e t e r m i n e d to b e resistant may n o t b e t h e organism responsible for t h e infection. In diseases s u c h as o r o p h a r y n g e a l Candida infections, species s u c h as C.glabrata a n d C. krusei are often isolated from oral samples b u t t h e r e is d o u b t w h e t h e r t h e y always c o n t r i b u t e to s y m p t o m a t i c pathology. 22.23
Therapy deemed
n=
n=
Appropriate
431
81% (76-84)
725
64% (61-68)
CONCLUSIONS
inappropriate
172
63% (55-70)
167
56% (48-63)
179
61% (53-68)
T h e very t e r m ' b r e a k p o i n t ' c o n t r i b u t e s to t h e traditional image of antimicrobial susceptibility testing as a sharp-edged razor, capable of differentiating a n a p p r o p r i a t e from an i n a p p r o p r i a t e treatment. T h e reality, as e v i d e n c e d b y m a n y publications t h a t actively analyze t h e association b e t w e e n susceptibility in vitro a n d clinical t r e a t m e n t results, is t h a t susceptibility testing is p e r h a p s analogous to w e a t h e r forecasts. T h e m e t h o d s u s e d in w e a t h e r forecasting, as w i t h susceptibility testing, are o f t e n b a s e d o n sophisticated technology.They p r o v i d e a n excellent statistical basis for general w a r n i n g s of adverse events b u t t h e y c a n n o t accurately predict t e m p e r a t u r e or precipitation in any specific location at any specific time. In clinical practice it is i m p o r t a n t for physicians to a c k n o w l e d g e that t h e r a p e u t i c success w i t h fungal diseases is partly or largely a f u n c t i o n of t h e i m m u n e status of t h e patient, t h e nature of t h e underlying disease, a n d o t h e r factors u n r e l a t e d to susceptibility of t h e infecting a g e n t in vitro. 1°'2~25A laboratory finding of resistance should b e a w a r n i n g for t h e clinician, b u t n o t an i m m e d i a t e contraindication for a particular agent.
None
Clinical setting
Reference
bacteremia
Gram-negative bacteremia
16
17
*"Appropriate" therapy means use of an agent for which the bacterial isolate was susceptible in vitro, or for which the isolated species is usually found susceptible and which could be given to achieve sufficient blood or tissue concentrations in the given case of infection. *95% confidence intervals determined by binomial approximation.
association b e t w e e n microbial resistance a n d t r e a t m e n t failure is b e t t e r for s o m e t y p e s of i n f e c t i o n t h a n for others, a n d it is b e t t e r for s o m e m i c r o b i a l species t h a n for others. T h o r n s b e r r y et a115 b r o k e d o w n t h e i r large d a t a b a s e for c e f o t a x i m e MICs versus t r e a t m e n t o u t c o m e b y different bacterial g r o u p s ( t h e data in Table 2 s h o w t h e overall results). For Gram-negative bacilli o t h e r t h a n Enterob a c t e r i a c e a e a n d Pseudomonas spp., for example, t h e y f o u n d a h i g h c o r r e l a t i o n b e t w e e n susceptibility in vitro a n d good t r e a t m e n t o u t c o m e s , w i t h e r a d i c a t i o n of t h e b a c t e r i a in 98% of p a t i e n t s i n f e c t e d w i t h s u s c e p t i b l e isolates. However, e v e n for t h e s e organisms, 50% e r a d i c a t i o n was d o c u m e n t e d for t h e p a t i e n t s i n f e c t e d w i t h isolates resistant to c e f o t a x i m e in vitro. For t h e e n t e r o c o c c i as a group, t h e clinical e r a d i c a t i o n rate for 'resistant' isolates w a s actually h i g h e r t h a n for ' s u s c e p t i b l e ' isolates (82% versus 65%), b u t this result reflects a relatively l o w n u m b e r of resistant isolates, as is also s e e n in Table 2 for Pseudomonas spp. in .the data f r o m Flick a n d Cluff. 13 In t h e i r large study of c a n d i d e m i a , Rex et al 7 f o u n d an. inverse c o r r e l a t i o n b e t w e e n antifungal susceptibility test results a n d clinical t r e a t m e n t success (this study i n v o l v e d a m p h o t e r i c i n B as well as fluconazole). In t h e same way as reasons c a n b e postulated for treatm e n t failures w i t h susceptible organisms, t r e a t m e n t successes w i t h resistant ones m i g h t b e e x p l a i n e d in several ways. Subinhibitory effects of amimicrobial agents m i g h t tip t h e balance in favor of t h e host. For b o t h antibacterial agents 18,19 a n d a n antifungal agent, 2° s u b i n h i b i t o r y c o n c e n t r a t i o n s of inhibitors have b e e n s h o w n to alter microbial e x p r e s s i o n of toxins or surface molectfles that may b e involved in t h e infective process. An agent may interact synergistically w i t h a host m o l e c u l e a n d thus eradicate an o t h e r w i s e resistant organism: s u c h effects have b e e n d e s c r i b e d at least for g e n t a m i c i n a n d h u m a n amniotic fluid. ~1 Finally, in some
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References I. National Committee for Clinical Laboratory Standards. Reference method for broth dilution antifungal susceptibility testing of yeasts.Approved standard M27-A. NCCL5 1997. Villanova, PA. 2. Moore RD, Lietman PS, Smith CR. Clinical response to aminoglycoside therapy: importance of the ratio of peak concentration to minimal inhibitory concentration. J Infect Dis 1987; 155:93-99. 3. Amsterdam D. Susceptibility testing of antimicrobials in liquid media. In: LorianV (ed).Antibiotics in Laboratory Medicine, 4th edn. Baltimore: William & Wilkins, 1996: 52- I I I. 4. Greenwood D. In vitro veritas. Antimicrobial susceptibility tests and their clinical relevance.J Infect Dis 1981; 144: 380-385. 5. Barchiesi F,Hollis RJ, Mcgough DA, Scalise G, Rinaldi MG, Pfaller MA. DNA subtypes and fluconazole susceptibilities of Candida albicans isolates from the oral cavities of patients with AIDS. Clin Infect Dis 1995; 20: 634-640. 6. Espinel-lngroffA, Rodriguez-Tudela JL, Martinez-Suarez JV. Comparison of two alternative microdilution procedures with the National Committee for Clinical Laboratory Standards reference macrodilution method M27-P for in vitro testing of fluconazole-resistant and -susceptible isolates of Candida albicans. J Clin Microbiol 1995; 33:3154-3158.
7. Rex JH, Pfaller MA, BarryAL, Nelson PW, Webb CD. Antifungal susceptibility testing of isolates from a randomized, multicenter trial of fluconazole versus amphotericin B as treatment of nonneutropenic patients with candidemia.Antimicrob Agents Chemother 1995; 39: 40-44. 8. Phillips R Zemcov J, Mahmood W, Montaner JSG, Craib K, Clarke AM. Itraconazole cyclodextrin solution for tquconazole refractory oropharyngeal candidiasis in AIDS: correlation of clinical response with in vitro susceptibility.AIDS 1996; I 0:1369-1376. 9. Quereda C, Polanco AM, Giner C et al. Correlation between in vitro resistance to fluconazole and clinical outcome of oropharyngeal candidiasis in HIV-infected patients. Eur J Clin Microbiol Infect Dis 1996; 15: 30-37. 10. Rex JH, Pfaller MA, Galgiani JN et al. Development of interpretive breakpoints for antifungal susceptibility testing: conceptual framework and analysis of in vitro-in vivo correlation data for fluconazole, itraconazole and Candida infections. Clin Infect Dis 1997; 24: 235-247. I I. Johnson CC. In vitro testing: correlations of bacterial susceptibility, body fluid levels, and effectiveness of antibacterial therapy. In: Lorian V (ed).Antibiotics in Laboratory Medicine, 4th edn. Baltimore: William & Wilkins, 1996:813-834. 12. Lorian V, Burns L, Ernst J. Predictive value of susceptibility tests for the outcome of antibacterial therapy.J Antimicrob Chemother 1990; 25:175-18 I. 13. Flick MR, Cluff LE. Pseudomonas bacteremia: review of 108 cases. Am J Med 1976; 60:501-508. 14. GerberAU, CraigWA.Worldwide clinical experience with cefoperazone. Drugs 198 I; 22 (Suppl I): 108-I 18. 15. Thornsberry C,Jones RN, BarryAL, Fuchs PC.Antimicrobial susceptibility tests with cefotaxime and correlation with clinical bacteriologic response. Rev Infect Dis 1982; 4 (Suppl): $316-$324.
16. Kreger BE, Craven DE, McCabe WR. Gram-negative bacteremia. IV. Re-evaluation of clinical features and treatment in 612 patients. Am J Med 1980; 68: 344-354. 17. Bryan CS, Reynolds KL, Brenner ER.Analysis of I 186 episodes of gram-negative bacteremia in non-university hospitals: the effects of antimicrobial therapy. Rev Infect Dis 1983; 5: 629-638. 18. Dupont MJ, Lapointe JR. Effects of Pseudomonas aeruginosa alginate expression of direct plating and culture of fresh cystic fibrosis sputum on to Pseudomonas isolation agar containing subinhibitory concentrations of roxithromycin and rifampicin.J Antimicrob Chemother 1995; 36:231-236. 19. HostackaA. Postantibiotic effects of subinhibitory concentrations of some antibiotics and their influence on Pseudomonas aeruginosa enzymic activity. Folia Microbiol 1996; 4 I: 39-42. 20. Angiolella L, Facchin M, Stringaro A, Maras B, Simonetti N, Cassone A. Identification of a glucan-associated enolase as a main cell wall protein of Candida albicans and an indirect target of lipopeptide antimycotics.J Infect Dis 1996; 173: 684-690. 2 I. Miglioli PA, Schoffel U, Gianfranceschi L. The in vitro synergistic inhibitory effect of human amniotic fluid and gentamicin on growth of Escherichia coli. Chemotherapy 1996; 42: 206-209. 22. Maenza JR, MerzWG, Romagnoli MJ, Keruly JC, Moore RC, Gallant JE. Infection due to fluconazole-resistant Candida in patients with AIDS: prevalence and microbiology. Clin Infect Dis 1997; 24: 28-34. 23. Dronda F,Alonsosanz M, Laguna F et al. Mixed oropharyngeal candidiasis due to Candida albicans and non-albicans Candida strains in HIV-infected patients.J Clin Microbiol Infect Dis 1996; 15:446-452. 24. Pittrow L, PenkA. Plasma and tissue concentrations of fluconazole and their correlation to breakpoints. Mycoses 1997;40:25-32. 25. Ghannoum MA, RexJH, GalgianiJN. Susceptibility testing of fungi: current status of correlation of in vitro data with clinical outcome. J Clin Microbiol 1996; 34: 489-495.
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