Eur J Clin Microbiol Infect Dis (2004) 23: 506–508 DOI 10.1007/s10096-004-1136-2
CONCISE ARTICLE
J. Chandenier . K. D. Adou-Bryn . C. Douchet . B. Sar . M. Kombila . D. Swinne . M. Thérizol-Ferly . Y. Buisson . D. Richard-Lenoble
In vitro activity of amphotericin B, fluconazole and voriconazole against 162 Cryptococcus neoformans isolates from Africa and Cambodia Published online: 13 May 2004 # Springer-Verlag 2004
Abstract In order to determine the potential role that various antifungal agents might have in the management of cryptococcosis in tropical areas, the in vitro susceptibility of Cryptococcus neoformans isolates from Africa (n=52) and Cambodia (n=110) to three antifungal agents (amphotericin B, fluconazole and voriconazole) were compared using the E-test method. The results of this study (i) confirm the value of the E-test for testing the in vitro susceptibility of C. neoformans towards voriconazole; (ii) provide the first evidence demonstrating good activity of amphotericin B, fluconazole and voriconazole against Cambodian isolates; and (iii) show there are differences in susceptibility between African and Asian C. neoformans isolates, with Cambodian isolates appearing less susceptible to the agents tested but with amphotericin B maintaining good activity.
Introduction Cryptococcosis still represents a major public health problem in Africa and Asia and its treatment remains J. Chandenier (*) . K. D. Adou-Bryn . C. Douchet . M. Thérizol-Ferly . D. Richard-Lenoble Service de Parasitologie-Mycologie-Médecine Tropicale, Centre Hospitalier et Universitaire, 37000 Tours, France e-mail:
[email protected] Tel.: +33-2-47475902 Fax: +33-2-47478082 B. Sar . Y. Buisson Institut Pasteur du Cambodge, Phnom Penh, Cambodia M. Kombila Département de Parasitologie-Mycologie-Médecine Tropicale, FMSS, BP 4009 Libreville, Gabon D. Swinne Institut Scientifique de Santé Publique-Mycologie, Wytsman Street, 141050 Brussels, Belgium
problematic [1]. The reference treatment, based mainly on amphotericin B (AMB), is difficult to implement in these underprivileged regions due to the necessity of administering the product by intravenous infusion over long periods, and under strict surveillance, in light of its renal toxicity. In this context, fluconazole (FLU) has represented a significant step forward in the treatment of patients, since it is not only effective, but also considerably simplifies the overall management of treatment and follow-up. Fifteen years after the commercialization of FLU in France, however, strains of Cryptococcus neoformans have been identified that are resistant to this azole, mainly in areas where the product has been used widely in curative treatment and in various primary and secondary prevention schemes [2]. It therefore appears important to continue the pharmacological and clinical development of new agents with potential activity against C. neoformans. Of the products currently available, voriconazole (VOR) appears to represent one of the most interesting alternatives [3, 4]. We therefore tested the in vitro efficacy of AMB, FLU and VOR against 52 African isolates and 110 Cambodian isolates of C. neoformans using the E-test (AB BIODISK, Solna, Sweden), in order to determine the potential role that these antifungal agents might have in the management of cryptococcosis in tropical areas.
Materials and methods All of the C. neoformans isolates included in this study were obtained from the cerebrospinal fluid of HIV-positive hospital inpatients. Only one isolate was taken from each patient, and under the circumstances, it is unlikely that the patients had received any previous treatment. The African strains were obtained from patients in the following countries: 19 from Rwanda, 19 from the Congo, nine from Gabon, and five from the Ivory Coast. They were collected between 1994 and 2001. The 110 Cambodian strains came from patients attending the Calmette Hospital in Phnom Penh and were collected between 1998 and 2001. All of the strains were transported to the Service de Parasitologie-Mycologie-Médecine Tropicale, Centre Hospitalier et Universitaire in Tours, France, for further testing.
507 The strains were all demonstrated using the India ink test. For each isolate, the species was confirmed by hydrolysis of urea, culture at 37°C, agglutination using polyclonal antiserum for Cryptococcus spp. and by the brown appearance of colonies on modified Pal’s medium [5]. Finally, the serotypes were determined using a second agglutination test carried out on slides using specific antisera prepared in our unit. Prior to testing, all strains had been stored in physiological saline or were freeze-dried. Before performing the in vitro tests, the strains were cultivated for 48 h on Pal’s medium. The E-tests were then performed and read according to the method recommended by the manufacturer. A suspension was prepared with a turbidity of 0.5 on the McFarland scale (roughly 5×105 yeasts/ml) by diluting colonies in 0.85% normal saline solution. This suspension was loaded onto Petri dishes with a diameter of 90 mm containing RPMI agar +2% glucose (AES-Laboratory, Combourg, France) using a sterile swab. The Etest strips (AMB and FLU: AES laboratory; VOR: Pfizer, New York, USA) were applied when the surface of the agar had recovered its initial appearance on drying. The plates were read after 72 h of incubation at 35°C. The MICs were read at 95% inhibition for AMB and 80% inhibition for the azoles. Control tests were regularly performed using Candida albicans ATCC 90028, Candida parapsilosis ATCC 22019 and Candida kruzei ATCC 6258 available in our laboratory.
Results and discussion All of the C. neoformans isolates were of serotype A. Susceptibility testing of the control strains C. albicans, C. parapsilosis and C. kruzei to the three antifungal agents gave results consistent with the known values for these strains. The results of susceptibility testing performed on the 162 C. neoformans isolates and the three antifungal agents are summarised in Table 1. All of the strains were highly sensitive to AMB, with a concentration of 0.094 μg/ml being sufficient to inhibit the growth of all isolates studied, except for one Cambodian strain for which the MIC was 0.125 μg/ml. However, when comparing the results for the African strains with those for the Cambodian strains, a difference of three gradations was noted on the E-test strips for the MIC50 values and six gradations for the MIC90 values, with higher values being noted for the Asian strains each time. Moreover, the MIC50 and MIC90 values of AMB for the African strains inhibited only 12 and 35% of the Cambodian strains, respectively (significant difference: P32 μg/ml for 1), and the MIC50 and MIC90 values were both higher by one gradation on the E-test strips for the Asian strains compared with the African strains.
Table 1 In vitro susceptibility of 162 Cryptococcus neoformans isolates to amphotericin B, fluconazole and voriconazole Antifungal agent
MIC in μg/ml MIC50
African isolates (n=52)a Amphotericin B 0.006 Fluconazole 8 Voriconazole 0.125 Cambodian isolates (n=110) Amphotericin B 0.016 Fluconazole 16 Voriconazole 0.19
MIC90
Range
0.012 32 0.25
256b 0.004–0.5
0.094 32 0.38
256c 0.012–>32d
a
Congo, 19 isolates; Rwanda, 19 isolates; Gabon, 9 isolates; Ivory Coast, 5 isolates b Three isolates ≥64 μg/ml c Seven isolates ≥64 μg/ml d One isolate >32 μg/ml
It is now accepted that in vitro chemosensitivity testing of yeasts can make a useful contribution to the management of antifungal treatment [6]. In fact, it appears that the E-test on RPMI-agar-glucose could replace the test recommended by the National Committee for Clinical Laboratory Standards for this purpose, which is particularly onerous to perform and not well-suited for studying a large number of strains [7]. Moreover, results comparable to those achieved using the National Committee for Clinical Laboratory Standards method have already been obtained using the E-test technique with AMB [8], FLU [9] and VOR [10] against C. neoformans. Against the African isolates in our study, AMB showed low MIC values that did not exceed 0.094 μg/ml for any of the strains. The MIC50 and MIC90 values for FLU (8 and 32 μg/ml, respectively) were identical to those observed in previous studies using strains from the USA [11], the UK [12], and Uganda in 1998 [12] and 1999 [2]. However, we also found high MIC values for this agent against some isolates (i.e. 64 μg/ml for 2 isolates and >256 μg/ml for 1 isolate). This phenomenon was not observed in the study conducted in 1999 [2], and no precise data on this subject was provided in 1998 [12]. Since azoles are still not widely used in these countries, our finding is probably attributable to innate resistance, as has been suggested previously [12]. Finally, our results show that VOR inhibited 50 and 90% of the African strains with MICs of 0.125 and 0.25 μg/ml, respectively, with the maximum MIC value being 0.5 μg/ml. These results are similar to those of the only other study conducted using this agent on a population of C. neoformans isolates from Africa (MIC50, 0.12 μg/ml; MIC90, 0.25 μg/ml), but that study utilized the NCCLS method [2]. The very low MICs for VOR found in our study are therefore encouraging in terms of the possible use of this agent in the treatment of patients infected with C. neoformans of African origin.
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We also determined the sensitivity profiles of C. neoformans isolates from Cambodia, a country where AIDS and its treatment is currently an acute problem [13]. Since the MICs of AMB did not exceed 0.125 μg/ml, this agent remains highly active against strains from this country. Although the MIC50 and MIC90 values of AMB were higher against the isolates from this country than those from Africa, none of the results reached the value of 2 μg/ml, which has been suggested as the resistance threshold for this agent [11]. For FLU, the MIC50 and MIC90 values of 16 and 32 μg/ml, respectively, found in our study are also comparable with the results of other studies conducted using strains from the USA [11], the UK [12], and Uganda [2]. However, the presence of seven (6.3%) isolates for which the MICs equalled or exceeded 64 μg/ml (including 5 for which MICs were >256 μg/ml) is more surprising, given that this result was obtained for only 1.5% (8 of 522) of C. neoformans isolates studied in the USA [11] and 0% in Uganda [2]. However, our finding is close to the 4.9–5.6% observed in the UK [12]. The percentage of high MIC results for FLU perhaps reflects a trend specific to Southeast Asia, since one of two previous studies [14, 15] conducted using C. neoformans strains from Thailand found a FLU MIC of 64 μg/ml for 20% of the strains tested [14]. For VOR, the MIC50 and MIC90 values of 0.19 and 0.38 μg/ml we obtained against the Cambodian isolates remain satisfactory. Although they are higher than the results achieved with the African isolates, the values are still close. In addition, the results we obtained for the Cambodian isolates and the African isolates differ by three gradations on the E-test strips from the figures obtained in the previous study of strains from the USA [2]. Finally, only one of our seven Cambodian isolates with reduced sensitivity to FLU also had reduced sensitivity to VOR. The results of this study appear to confirm that crossresistance of C. neoformans to the various azoles is not a constant phenomenon and that voriconazole could represent a useful treatment alternative for cryptococci that demonstrate resistance to FLU. Acknowledgements We thank Pfizer for kindly providing the voriconazole E-test strips and for help with the translation of this article. The 19 strains from Rwanda used in this study are available at “BCCM/IHEM biomedical fungi and yeasts collection, Scientific Institute of Public Health, Wytsan Street 14, 1050 Brussels, Belgium”.
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