Mycology
Comparison of Etests and Vitek 2® to broth microdilution for the susceptibility testing of Cryptococcus neoformans

https://doi.org/10.1016/j.diagmicrobio.2014.09.006Get rights and content

Highlights

  • Low MIC of antifungal drugs was exhibited by isolates of C. neoformans from Durban.

  • Vitek 2® compared well with broth microdilution (BMD) for fluconazole, amphotericin, and flucytosine.

  • Etests can be used instead of BMD for testing fluconazole and voriconazole.

  • Amphotericin B Etests were less reliable and gave lower MIC results than BMD.

  • MIC testing using flucytosine Etests cannot be recommended for C. neoformans.

Abstract

We determined the susceptibility of 102 clinical isolates Cryptococcus neoformans from Durban, South Africa, to amphotericin B, fluconazole, flucytosine, and voriconazole using broth microdilution (BMD) according to the Clinical and Laboratory Standards Institute M27-A3 document and compared these results with Etest and Vitek 2®. Essential agreement (EA) of Etest and Vitek 2® compared to BMD was determined. Low MICs that were below the epidemiological cutoff values of the 4 antifungal agents tested were demonstrated by all isolates. The EA of Etests for fluconazole, amphotericin, and voriconazole was 95.1%, 83.3%, and 91.2%, respectively, and for Vitek 2® EA for fluconazole, amphotericin, and flucytosine was 97.1%, 95.1%, and 97.1%, respectively. The Vitek 2® showed good agreement with BMD and is a suitable alternative. Etests demonstrated good EA for azoles only. Clinical cryptococcal isolates from Durban remain susceptible to current recommended antifungal therapy.

Introduction

Nearly a million cases of cryptococcal meningitis (CM) are estimated to occur globally each year, resulting in an estimated 624 700 deaths, the majority of which occur within 3 months of infection (Park et al., 2009). It is associated with a high early mortality rate and disease recurrence is not uncommon. With the HIV pandemic, CM has become the most common cause of adult meningitis in South Africa (Jarvis et al., 2010). Moreover, 44% of deaths due to HIV-related infections in South Africa are due to cryptococcosis (Corbett et al., 2002).

Combination therapy using amphotericin B with flucytosine is the superior regime for the induction treatment of CM, as it results in rapid sterilisation of cerebrospinal fluid (CSF); a lower relapse rate; and, importantly, lower mortality rates (Brouwer et al., 2004, Day et al., 2013, Van der Horst et al., 1997). Flucytosine is not available in South Africa, and the current recommendation is amphotericin B combined with fluconazole for induction followed by fluconazole monotherapy in the consolidation and maintenance phases of treatment (Govender et al., 2013). Not all patients achieve CSF cryptococcal sterility prior to the change to fluconazole therapy; therefore, cryptococcal susceptibility to this agent is critical for long-term clinical success. Indeed, induction CM therapy is reliant on high-dose fluconazole in resource-limited settings, where it is mostly used alone or in place of flucytosine, in combination with short-course amphotericin B.

Globally, cryptococcal resistance to amphotericin B, fluconazole, and flucytosine remains under 1%, and a study examining the antifungal susceptibility patterns on isolates collected worldwide across 5 regions, including Africa, over a 15-year interval did not show significant increase in resistance (Pfaller et al., 2005). Voriconazole is active against Cryptococcus neoformans, with low MICs demonstrated even in isolates, which show resistance to fluconazole (Pfaller et al., 1999). Anecdotally, voriconazole has been used with success to treat CM and may be used in cases that have failed conventional therapy (Perfect et al., 2003, Yong, 2011).

Antifungal susceptibility testing may be used to predict the outcome of treatment in patients with CM. High fluconazole MICs have been associated with therapeutic failure and relapse (Aller et al., 2000b, Bicanic et al., 2006, Friese et al., 2001, Lee et al., 2012, Witt et al., 1996). Furthermore, studies investigating serial isolates from patients presenting with CM relapses have demonstrated an increase in fluconazole MIC over time in some isolates (Arechavala et al., 2009, Brandt et al., 1996, Casadevall et al., 1993, Govender et al., 2011). Fluconazole has been used extensively without cost for the treatment of CM and esophageal candidiasis for several years in South Africa under the Pfizer donation programme, and this together with the recommendation of its use for pre-emptive therapy in this area, which has a high prevalence of CM, may fuel the development of resistance through selective pressure (Govender et al., 2013).

Although monitoring for the emergence of resistance to antifungal agents is critical in the management of cryptococcosis, antifungal susceptibility testing is not performed routinely.

The current method recommended by the Clinical and Laboratory Standards Institute (CLSI) and European Committee for Antimicrobial Susceptibility Testing (EUCAST) for antifungal susceptibility testing of yeasts is broth dilution (CLSI document M27-A3), which is both labour intensive and technically challenging (CLSI, 2008). Commercially available Etests (bioMérieux S.A., Marcy l’Etoile, France) and Vitek 2® (bioMérieux S.A.) offer a faster and simpler alternative. Although there are adequate data to support the use of these tests for Candida spp., data are limited on their utility for C. neoformans (Aller et al., 2000a, Cuenca-Estrella et al., 2010, González et al., 2010, Matar et al., 2003, Maxwell et al., 2003, Pfaller et al., 2007).

The aims of this study were to determine the antifungal susceptibility of C. neoformans clinical isolates from Durban, South Africa, to amphotericin B, flucytosine, fluconazole, and voriconazole using the reference CLSI M27-A3 broth microdilution (BMD) method and to compare these results with the commercially available Etests and Vitek 2®.

Section snippets

Patients and cryptococcal isolates

One hundred and twenty-eight HIV-infected patients experiencing their first episode of CM were enrolled prospectively into a longitudinal cohort study based at a tertiary hospital in Durban, South Africa, between August 2009 and March 2011. Patients who were 18 years or older who had a positive cryptococcal antigen test on CSF were included in the study. All patients were naïve to antifungal therapy and combination antiretroviral therapy at enrolment (Chang et al., 2013). Cryptococcal isolates

Results

One hundred and thirteen patients of the 128 enrolled into the study had CSF that was culture positive for C. neoformans. Eleven isolates were excluded from the analysis: 6 isolates grew poorly in the Vitek 2® growth control well and were terminated, 1 did not grow in the BMD growth control well or in the Vitek 2®, and 4 did not grow on RPMI media. One hundred and two isolates were used in the analysis. Etests for flucytosine were discontinued after the first batch of 10 isolates was tested

Discussion

Low MICs for amphotericin B, flucytosine, fluconazole, and voriconazole using BMD and the commercially available Etests and Vitek 2® were demonstrated by clinical isolates of C. neoformans from Durban, South Africa. Therefore, the current treatment regime used in South Africa of amphotericin B and fluconazole is still appropriate for therapy. The Vitek 2® compares very well with BMD with EA for fluconazole, amphotericin, and flucytosine of 97.1%, 95.1%, and 97.1%, respectively. The EA of Etests

Conflict of interest

Voriconazole powder was donated by Pfizer, but the company bore no influence nor was able to access the data and analysis. There was no other conflict of interest.

Acknowledgments

We would like to thank the study participants, the clinicians who conducted the clinical trial, and the staff from the microbiology laboratory at Inkosi Albert Luthuli Central Hospital, Durban. Funding was provided by the REACH initiative and the NHLS Research Trust. We would also like to thank Dr N. Govender who provided expert technical guidance with the methodology for BMD. Finally, we would like to thank Pfizer, Groton, Connecticut, for supplying voriconazole powder for BMD.

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