Methods of studying the in vitro antifungal susceptibility: what else?

AL WelcomeS. Picot

Abstract: 

Fungal infections often have a poor prognosis and wanderings of initial treatment sometimes participate in this dark evolution. Few fungicides drugs and the use of fungistatic molecule is an unstable balance between the therapeutic activity, medical and financial toxicity and the risk of escape. It is therefore legitimate to expect a technical in vitro prior brings pertinent arguments for choosing a treatment. Still, a solid method still missing and the answers often do not lead to a therapeutic relevance. However, pending a solution, it is possible to establish the circumstances in which the sensitivity tests in vitro are useful and a list of strengths and weaknesses of the available methods.

Why test the sensitivity of fungi to antifungal agents in vitro ?

The decision to perform a test in vitro should not be systematic, but based on a medical logic, mycological, epidemiological or experimental.

Medical logic: the main situation that leads a clinician to request a determination of the fungus antifungal susceptibility was the finding of a therapeutic failure despite adequate antifungal therapy. The identification of a fungal agent in a normally sterile site (blood, CSF, biopsies ...) may also justify a determination of sensitivity. An azole treatment history justifies the determination of the sensitivity of Candida because azole may induce resistance.

Mycological logic: mycologist may prompt the clinician to ask this determination when identifying mushrooms known to have reduced sensitivity to antifungal agents, including Candida glabrata to azoles or Candida parapsilosis for echinocandins.

Epidemiological Logic: studies in vitro for epidemiological purposes, to track the evolution of these sensitivities in time within a service, a hospital or a multicentre group. This evolution can sometimes incite change treatment practices. It is obvious that the use of these multicenter data implies that the evaluation methods are the same in the different centers.

Experimental logic: the screening of new antifungal molecules used to determine the sensitivities in vitro and allows confirmation of new antifungal candidates. Using techniques chessboard could meet better the questions posed by the fungal associations, in particular the study of their antagonistic or synergistic effects.

The choice of method: The choice is difficult, given the technical panel available and their respective limits. It should be noted that performance may be different depending fungi (including filamentous fungi) or antifungal agents (eg 5-fluorocytosine).

There are two reference techniques proposed by the CLSI (Clinical and Laboratory Standards Institute) and EUCAST (European Committee on Antimicrobial Susceptibility Testing), based on the principle of macro and micro dilution of the liquid medium in antifungal and for determining yeast sensitivities or filamentous fungi. A CLSI method of agar diffusion disk is also available for yeast and being developed by EUCAST. These techniques called reference are not applicable in daily practice outside expert centers. This opens the door to the provision of more practical techniques, but with limitations.

The main techniques available are:

- E-test® (Biomerieux): medium agar diffusion method using strips impregnated antifungal gradients

- NeoSensitabsÒ (Rosco): medium agar diffusion method using disks

- Sensititre YeastOneÒ (Trek Diagnostic System) and FungitestÒ (Sanofi Pasteur) microtiter plates ready for use, the principle is based on liquid-based reference technique

- ATBFungus 3® (Biomerieux) microtiter galleries ready-whose principle is based on liquid-based reference technique

Reading rule is the determination of the value of the minimal inhibitory concentration or MIC to the first break of growth except for amphotericin b is strictly fungicide. Difficulty in reading the tests can be met, with the azole particular, because there is a phenomenon of train which results in a regrowth of colonies within the ellipse of inhibition. The results of these techniques are expressed in MIC. Reference breakpoints are mainly limited toCandida and Aspergillus for azoles, 5-and flurorocytosine amphotericin b, and depend techniques. Work is underway to establish these breakpoints vis-à-vis the echinocandins, caspofungin particular.

The main issue with these techniques is the significance of the values ​​obtained in vitro as part of the fungal disease. The predictive value of these tests is far from clear, with the notable exception of oropharyngeal candidiasis treated with fluconazole. Besides the lack of data on the correlation clinicobiological, other parameters involved in the effectiveness of an antifungal, the pharmacokinetic parameters. When determining a decreased sensitivity before starting treatment, the reasonable attitude is to avoid this antifungal. However, an antifungal change should not be recommended if the patient is improving treatment when it is shown that the fungal agent has decreased sensitivity in vitro .

What are the locks open to move towards a consensus?

Standardization efforts are still required to enable the establishment of longitudinal studies and epidemiological vision. The use of internal quality control (reference strains) is recommended as with any technique, but especially the establishment of an external quality control system is highly necessary for portability of results between centers.

The implementation of the STANDARD ISO / WD 11764 (laboratory test systems - Susceptibility testing of fungi) certainly contribute to the success of this standardization.

However, the relevance in vivo of these techniques is a debate that is timely. The interpretation of fungi susceptibility results in vitro is difficult and requires a dialogue between the mycologist and clinician, specific to each clinical situation, to give meaning to biological result. New methods have been described in recent years, such as microarrays and flow cytometry without clear applications in medical practice have already been published.

2009

Full conference title: 

Réunion Interdisciplinaire de Chimiothérapie Anti Infectieuse
    • RICAI 29th (2009)