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Susceptibility testing and models for Aspergillus

Overview of methodology

At least 18 methodologies have been employed for susceptibility testing of Aspergillus and there has been no rigorous attempt to compare one methodology with another (Denning et al, 1992).

Media employed have included yeast nitrogen broth with 0.5% glucose, synthetic amino acid medium/fungi, minimal essential medium, antibiotic medium 3, Rowley-Huber broth, casitone complex broth, yeast morphology agar and brain-heart infusion agar. The pH range has been from 4.0 to 7.4 and temperature of incu

Most tests have been done without agitation, which might increase oxygenation.

The majority of tests have been read at 1 and 3 days. The end-point determination has usually been a visual one but this has been variable.

A few laboratories have also determined minimum fungicidal concentrations in addition to inhibitory concentrations and this has also been done in a variety of ways.

The result of this diversity of methodology has been a diversity in test results. Some test methods yielded a very narrow range of results which is unlikely to be a genuine reflection of the organism's susceptibility to the antifungal used, given the range of clinical responses that have been noted to all of the active drugs. In one large US study of itraconazole therapy for invasive aspergillosis, 50 isolates were susceptibility tested in one laboratory and all were apparently susceptible (Denning et al, 1992).A correlation with clinical outcome was therefore not possible.

On the other hand, some of the data is consistent with clinical experience. Most isolates have high MICs to flucytosine tested by many methods which is consistent with the minor efficacy of flucytosine in the therapy of invasive aspergillosis (Denning et al, 1992). Likewise many of the methods in which Aspergillus was tested against ketoconazole (which has no useful clinical activity for invasive aspergillosis) yielded high MICs (Denning et al, 1992). The converse was true with itraconazole in which the majority of test methods yielded susceptible isolates (Denning et al,1992). Thus susceptibility testing may be useful in testing a population of isolates but it is not yet possible to use the results from an individual isolate to guide therapy in a given patient.

There are various other difficulties associated with susceptibility testing of moulds. In our experience the two particularly difficult problems are end-point determination and test reproducibility with itraconazole. The end-point determination is difficult because the organism grows in a variety of different ways, sometimes at the meniscus, sometimes attached to the wall of the tube and sometimes free-floating.

In 1995 the National Committee for Clinical Laboratory Standards (NCCLS) Subcommittee on Antifungal Susceptibility Testing started work on the in vitro susceptibility testing of filamentous fungi including Aspergillus species by adapting the proposed NCCLS reference method for yeasts (Espinel-Ingroff et al 1995). Preliminary studies focussed on the reproducibility of the assay and no attempt was made to correlate the MIC values with clinical response to therapy. A broth microdilution method which showed good laboratory reproducibility for Aspergillus species and other filamentous moulds was proposed as a reference method (Espinel-Ingroff et al. 1997).

Amphotericin B

Clinical failure is frequent with amphotericin B therapy in patients with invasive aspergillosis. MICs to amphotericin B are in a narrow range with most testing systems, typically from 0.5-4 mcg/ml.

Recent work with carefully selected isolates from patients that received early appropriate amphotericin B therapy and yet failed therapy has revealed that in vitro testing fails to identify resistance when it can be demonstrated in vivo in an animal model (Verweij et al, 1998). The frequency of this phenomenon is unclear as yet and strenuous efforts are underway to develop an in vitro testing system that can detect resistance. The mechanism(s) of resistance is unclear.

Itraconazole and new azoles

In vitro resistance of A.fumigatus to itraconazole (confirmed in an animal model) has just been describe (Denning et al, 1997; Denning et al, 1997), although the frequency of resistance is probably low.

Resistance has been identified in isolates from the UK, US and Sweden. Different degrees of susceptibility to itraconazole have been found for different Aspergillus species, A. flavus and A. terreus being generally more susceptible in vitro than other species. Given the development of resistance, a more aggressive approach to obtaining positive cultures of Aspergillus for species-specific identification and susceptibility testing is appropriate.

A degree of cross resistance has been shown to the experimental azole SCH-56592 in vitro which has been confirmd in an animal model.In contrast, no in vivo cross resistance was demonstrated to voriconazole has been found and this finding has also been demonstrated (Denning, unpublished).

Combinations

Antifungal combinations against Aspergillus have been examined in few studies (Denning et al, 1994). Such data that exist are shown in the table but readers should again recognise that although a single method was used in these studies, the methodology for the determination of amphotericin B MICs is not validated and therefore combination work is also open to question.

The combination of amphotericin B and flucytosine was synergistic against 13% of tested isolates and an equal number showed antagonism. If this finding could be validated clinically there would be a strong case for routine synergy testing of this combination in patients treated with both agents.

A combination of amphotericin B and rifampicin showed synergy in the vast majority of isolates tested in two studies. Often synergy was profound but no antagonism was seen. There are some disadvantages to the use of rifampicin (mainly unfavourable drug interactions) but this in vitro data supports combination therapy.

Insufficient data is present regarding the combination of amphotericin B and itraconazole to evaluate in vitro synergy or otherwise. Ketoconazole and amphotericin B in combination impair the killing of Aspergillus in vitro. Surprisingly, azithromycin appears to be synergistic with amphotericin B against Aspergillus (Nguyen, 1997).

Antifungal combinations (Denning et al, 1992)

S = synergy/additive, I = indifferent, A = antagonistic

Professor Paul Verweij
Department of Medical Microbiology 
PO Box 9101
6500 HB Nijmegen
The Netherlands
T: +31 24 3614356
F: +31 24 3540339
P.Verweij@mmb.umcn.nl

David W. Denning FRCP FRCPath FIDSA FMedSci
Professor of Medicine and Medical Mycology
Director, National Aspergillosis Centre
Education and Research Centre
University Hospital of South Manchester (Wythenshawe Hospital)
Southmoor Road
Manchester M23 9LT UK

April 1998