In vitro susceptibility of Cryptococcus neoformans clinical isolates from Egypt to seven antifungal drugs

The in vitro susceptibility of 29 clinical isolates of Cryptococcus neoformans to fluconazole, miconazole, itraconazole, ketoconazole, flucytosine, nystatin and amphotericin B was tested by broth and colorimetric microdilution methods. Most of the isolates showed uniform patterns of susceptibility to the used antifungal agents. Only three isolates exhibited resistance [fourfold or greater rise in the minimum inhibitory concentrations (MICs)] to the tested antifungal drugs. The MIC50 and MIC90 were 0.5-8 mg l(-1) for 5-flucytosine, 0.2-8.25 mg l(-1) for nystatin, 0.5-16 mg l(-1) for fluconazole and 0.2-12.5 mg l(-1) for miconazole. However, MIC50 and MIC90 were in narrow range for the clinical yeast isolates in both methods used and showed 0.5-1 mg l(-1) for amphotericin B and 0.016-0.25 mg l(-1) for both ketoconazole and itraconazole. The combination of fluconazole plus flucytosine showed greater synergistic and fungicidal activity compared with that of fluconazole plus amphotericin B or the use of individual drugs.     

First isolation of Cryptococcus neoformans var. gattii from a native jungle tree in the Brazilian Amazon rainforest

Cryptococcus neoformans var. gattii was isolated for the first time from decaying wood in a hollow of a native jungle tree Guettarda acreana, in a wild area of an Amazon rainforest island, in Brazil. The presence of this variety in a virgin environment without either anthropic action or introduced vegetation is discussed with regard to the common knowledge of Cr. neoformans ecology.     

Study of Cryptococcus neoformans Varieties

Summary: We recommend L-canavanine-glycine bromothymol blue medium to differentiate C. neoformans var. neoformans from C. neoformans var. gattii. The former was isolated both from saprophytic and parasitic sources, the latter variety (7 isolates) being only isolated from clinical cases. One strain came from China, and six from Zaïre where they were isolated before 1969.
Zusammenfassung: Zur Unterscheidung zwischen C. neoformans var. neoformans und C. neoformans var. gattii wird das L-Canavanine-Glycin-Bromothymolblau-Medium empfohlen. C. neoformans var. gattii wurde nur von klinischen Fällen isoliert: ein Stamm aus China und sechs Stämme aus Zaïre. Alle Stämme aus Zaïre wurden vor 1969 isoliert.     

Müeller–Hinton methylene blue media as an alternative to RPMI 1640 for determining the susceptibility of Cryptococcus neoformans and Cryptococcus gattii to posaconazole with Etest

Mueller–Hinton modified agar (MH‐GMB) was compared with RPMI + 2% glucose–agar to determine the MICs of 80 isolates of Cryptococcus neoformans and C. gattii to posaconazole with Etest. MH‐GMB minimised trailing and agreement between both media was 94%. Agreement of M27‐A2 microbroth reference method was 98% with RPMI and 94% with MB‐GMB.     

Effects of several antifungal drug combinations against clinical and environmental isolates of Cryptococcus neoformans from China

The in vitro interactions of caspofungin (CSP) with terbinafine (TRB) and ravuconazole (RVC) with 5-fluorocytosine (5-FC) were tested against 82 clinical and environmental isolates of Cryptococcus neoformans from China. The interaction of CSP with TRB proved synergistic against those isolates with a CSP MIC < or =2 microg ml-1 (5% of the isolates), additive against 42% of the isolates and indifferent against 53%. The effects of RVC with 5-FC were synergistic, additive or indifferent against 8%, 26% and 67% of the isolates, respectively. No antagonistic effects were found among any of the drugs. The combinations of CSP with TRB and RVC with 5-FC may display beneficial effects in a strain-dependent manner, while in no case showed antagonistic effects. These data might be of use to design safer and more efficient treatments for patients with cryptococcosis and warrant further evaluation.     

Antifungal susceptibility of clinical and environmental isolates of Cryptococcus neoformans to four antifungal drugs determined by two techniques

A total of 64 Cryptococcus neoformans strains, including clinical and environmental Brazilian isolates var. neoformans and var. gattii, were tested for susceptibility to amphotericin B, 5-flucytosine, fluconazole and itraconazole. The tests were performed according to the recommendations of National Committee of Clinical Laboratory Standards and the method of macrodilution in liquid medium of Shadomy et al. [Manual de Microbiologia Clínica, 4th ed. Buenos Aires: Editorial Medica Panamericana, 1987: 1229-38]. For most drugs there was a significant difference between the readings taken at 24 and 48 h with both methods. When the minimum inhibitory concentrations obtained by the two techniques were compared, significant differences were observed for amphotericin B and fluconazole. Overall, differences in drug susceptibility with respect to the origin of the isolates or the variety of the fungus were not observed. As an exception, the gattii variety exhibited a high resistance rate to amphotericin B when the technique of Shadomy et al. was applied, a fact possibly related to the greater difficulty for treatment of the disease caused by this fungal variety.     

Cryptococcus neoformans var. neoformans and Munia Birds Cryptococcus neofovmans var. neoformans bei Prachtfinken

Cr. neoformans var. neoformans has been persistently demonstrated in the saprobic environment of caged munia birds at a local zoological garden. The pathogen was also cultured from the beaks, feathers and legs of these birds. It is suggested that the role of munia birds should be further investigated to understand the ecology of this important human pathogen.     

Feline meningitis due to Cryptococcus neoformans var. neoformans and review of feline cryptococcosis

Summary. Cryptococcus neoformans var. neoformans was ascertained as the prime cause of fatal meningitis in a young-adult female domestic cat who was suspected of rabies. The pathogen was isolated in heavy growth from the infected brain specimen on simplified sunflower seed ( Helianthus an-nuus ) medium at 30 C. Periodic acid-Schiff stained impression smear revealed numerous cryptococcal cells. Histologically, pia-arachnoid showed thickening along with many circular yeast cells with and without budding morphologically consistent with Cr. neoformans . The detailed typing of the strain indicated that it belonged to serotype AD and Filobasidiella neoformans var. neoformans mating type 'α'. The emphasis is given on early diagnosis by immunological and mycological techniques and prompt chemotherapy to avert the fatal consequences of this enigmatic mycosis.
Zusammenfassung. Cryptococcus neoformans var. neoformans wurde als Erreger einer tödlichen Meningitis einer jung erwachsenen weiblichen Hauskatze mit Tollwutverdacht nachgewiesen. Der Pilz lieiß sich mit reichem Wachstum aus dem infi-zierten Gehirn auf vereinfachtem Helianthus an-nuus -Samen-Agar bei 30 C anzüchten. PAS-ge-färbte Quetschpräparate zeigten zahlreiche Cryp-tococcus -Zellen. Histologisch war die Hirnhaut im Arachnoidalbereich verdickt mit vielen kugeligen Hefezellen mit und ohne Knospungen, die morpho-logisch mit Cr. neoformans vereinbar waren. Die Stammtypisierung ergab die Zugehörigkeit zum Se-rotyp AD und zum Mating-Typ a von Filobasi-diella neoformans var. neoformans . Die Notwendig-keit der Frühdiagnose durch immunologische und mykologische Techniken und des frühen Therapie-beginns wird betont, urn die fatalen Folgen dieser oft übersehenen Mykose abzuwenden.     

Isolation of Cryptococcus neoformans var. neoformans from canine otitis

Cryptococcus neoformans was demonstrated in the clinical material of a 15-year-old male dog with a history of chronic otitis externa by employing Helianthus annuus seed agar as a selective medium. The examination of the isolate for sexual compatibility on modified Helianthus annuus seed medium revealed that it belonged to Filobasidiella neoformans var. neoformans 'alpha' mating type. In the authors' view, this appears to be the first report of isolation of Cr. neoformans var. neoformans from diseased ear of a dog.     

Detection of Cryptococcus neoformans var. grubii in honeybee (Apis mellifera) colonies

The plant flora has an important role in the ecology of Cryptococcus neoformans. It is estimated that the environmental spreading and contamination of human beings with this yeast occurs via contaminated particles of plants. Cultivation of canopy parts of plants in selective media is the most widely used isolation method of this yeast. Cryptococcus neoformans var. grubii was isolated from honeybee colonies in Eucalyptus forests but was not isolated from the places where this flora did not exist. Our results indicate that the occurrence of C. neoformans in honeybee colonies during the flowering season of Eucalyptus spp. trees can be an important bioindicator for environmental yeast presence. The screening of honeybee colonies is a practical and a rapid method for the monitoring of the C. neoformans presence in flowering plants.     

Molecular epidemiology and in vitro antifungal susceptibility testing of 108 clinical Cryptococcus neoformans sensu lato and Cryptococcus gattii sensu lato isolates from Denmark

Cryptococcosis is mainly caused by members of the Cryptococcus gattii/Cryptococcus neoformans species complexes. Here, we report the molecular characterisation and in vitro antifungal susceptibility of Danish clinical cryptococcal isolates. Species, genotype, serotype and mating type were determined by amplified fragment length polymorphism (AFLP) fingerprinting and qPCR. EUCAST E.Def 7.2 MICs were determined for amphotericin B, flucytosine, fluconazole, voriconazole and isavuconazole. Most isolates were C. neoformans (serotype A; n = 66) and belonged to genotype AFLP1/VNI (n = 61) or AFLP1B/VNII (n = 5) followed by Cryptococcus deneoformans (serotype D; genotype AFLP2, n = 20), C. neoformans × C. deneoformans hybrids (serotype AD; genotype AFLP3, n = 13) and Cryptococcus curvatus (n = 2). Six isolates were C. gattii sensu lato, and one isolate was a C. deneoformans × C. gattii hybrid (genotype AFLP8). All isolates were amphotericin B susceptible. Flucytosine susceptibility was uniform MIC₅₀ of 4–8 mg l^?1 except for C. curvatus (MICs >32 mg l^?1). Cryptococcus gattii sensu lato isolates were somewhat less susceptible to the azoles. MICs of fluconazole (>32 mg l^?1), voriconazole (≥0.5 mg l^?1) and isavuconazole (0.06 and 0.25 mg l^?1 respectively) were elevated compared to the wild‐type population for 1/19 C. deneoformans and 1/2 C. curvatus isolates. Flucytosine MIC was elevated for 1/61 C. neoformans (>32 mg l^?1). Antifungal susceptibility revealed species‐specific differential susceptibility, but suggested acquired resistance was an infrequent phenomenon.     

Enzymatic characterisation of clinical isolates of Cryptococcus neoformans, Cryptococcus gattii and other environmental Cryptococcus spp.

This study compared the enzymatic activity of clinical isolates of Cryptococcus neoformans , Cryptococcus gattii , environmental isolates of C. neoformans and non-neoformans Cryptococcus . Most of the cryptococcal isolates investigated in this study exhibited proteinase and phospholipase activities. Laccase activity was detected from all the C. neoformans and C. gattii isolates, but not from the non-neoformans Cryptococcus isolates. There was no significant difference in the proteinase, phospholipase and laccase activities of C. neoformans and C. gattii . However, significant difference in the enzymatic activities of β-glucuronidase, α-glucosidase, β-glucosidase and N-acetyl-β-glucosaminidase between C. neoformans and C. gattii isolates was observed in this study. Environmental isolates of C. neoformans exhibited similar enzymatic profiles as the clinical isolates of C. neoformans , except for lower proteinase and laccase activities.     

In vitro susceptibility of Cryptococcus neoformans serotypes to GM 237354 derivative of the sordarin class

Zusammenfassung.   An 190 klinischen Isolaten von Cryptococcus neoformans aus unterschiedlichen Regionen Spaniens und Südamerikas wurde die Empfindlichkeit in vitro für das Sordanin-Derivat GM 237354 im Vergleich zu Amphotericin B getestet. Die MHK-Werte wurden mittels NCCLS-Mikroverdünnungs-Referenzmethode erhoben und ein Vergleich nach Cr . neoformans -Serotypen durchgeführt. Die MHKs für Amphotericin B lagen unter 1.0 µg ml⁻¹ (MHK_90% 0.5  μ g ml⁻¹, MHK_50% 0.125  μ g ml), aber fünf Isolate zeigten MHKs von 2.0  μ g ml⁻¹ für GM 237354 (MHK_90% 1.0  μ g ml⁻¹, MHK_50% 0.5  μ g ml⁻¹). Cryptococcus neoformans var. gatti Serotyp. B war signifikant weniger empfindlich als die Serotypen A und AD ( P =0.047 bzw. P =0.022).     

Comparison of Broth Dilution and Semisolid Agar Dilution for In Vitro Susceptibility Testing of Cryptococcus neoformans

Summary

We compared the in vitro activity of amphotericin B, flucytosine, itraconazole, fluconazole, ketoconazole and miconazole against 18 strains of Cryptococcus neoformans by using two methods: microbroth dilution and semisolid agar dilution.

By both of the methods minimum inhibitory concentrations (MICs) showed a wide range for all antifungal agents but not for amphotericin B. Statistically significant differences between the two methods were observed only with amphotericin B and flucytosine, p = 0.048 and p = 0.045 respectively.

Our study suggests that azole susceptibility testing for C. neoformans may be performed by the broth microdilution as well as the semisolid agar test. The choice of the method when testing amphotericin B and flucytosine is more problematic.     

Molecular typing and antifungal susceptibility of clinical and environmental Cryptococcus neoformans species complex isolates in Goiania, Brazil

A total of 124 Cryptococcus isolates, including 84 clinical strains obtained from cerebrospinal fluid from AIDS patients and 40 environmental isolates from pigeon excreta and from Eucalyptus trees, were studied. The varieties, serotypes, phospholipase activity and molecular profile of these isolates were determined. Cryptococcus neoformans var. grubii serotype A was identified in 120 isolates and Cryptococcus gattii serotype B in four isolates. The clinical isolates showed higher phospholipase activity than environmental isolates. Similar patterns of in vitro susceptibility to amphotericin B, fluconazole, itraconazole and voriconazole and no resistance were found for all isolates. Molecular type VNI ( C. neoformans var. grubii ) was recovered in 80 clinical and 40 environmental isolates while the type VGIII ( C. gattii ) was found in four clinical isolates. This study demonstrated for the first time the molecular types of clinical and environmental Cryptococcus isolates in the midwest Brazil region.     

Antifungal susceptibilities of Cryptococcus neoformans cerebrospinal fluid isolates from AIDS patients in Kenya

Poor susceptibility of Cryptococcus neoformans to fluconazole (FLC) is a matter of concern among clinicians in Africa. The emergence of resistance to FLC was recently reported in Kenya, but it is not known whether it is widespread. Thus, there is need for more antifungal drug susceptibility studies in Kenya. The aim of this study was to measure the in vitro antifungal drug susceptibilities of incident C. neoformans isolates from acquired immunodeficiency syndrome patients in Kenya. Antifungal susceptibility testing was performed in 67 C. neoformans isolates by broth microdilution method as outlined in the Clinical and Laboratory Standards Institute document M27-A3 using FLC, amphotericin B (AMB), voriconazole (VOR), ravuconazole (RAV) and flucytosine (5-FC). Isolates were grown on l-canavanine glycine bromothymol blue medium for serotype identification. Six per cent of the isolates were identified as C. neoformans var. gattii serotype B or C and 94% as C. neoformans var. neoformans. All isolates tested were susceptible to AMB, VOR and RAV (100%), and high susceptibilities were seen to FLC (97%), and 5-FC (90%). Only 3% and 10% of the isolates' susceptibility to FLC and 5-FC, respectively, was dose-dependent or intermediate. These results demonstrate high susceptibilities of incident C. neoformans isolates to FLC and AMB, antifungals used for treatment of cryptococcal meningitis in Kenya.     

Isolation of Cryptococcus species including C. neoformans from cloaca of pigeons

Upper digestive tract of the pigeon (Columba livia) is well known as a reservoir for different species of Cryptococcus, but lower portions are not so frequently studied. In the present study, we tested on selective media a total of 331 pigeon cloacal swabs; Cryptococcus spp. were recovered from 26 (7.85%). Cryptococcus uniguttulatus was isolated from 11 samples (3.32%), C. laurentii from six (1.81%), C. neoformans var. neoformans from six (1.81%) and C. albidus from three of them (0.91%). The results show the importance of pigeon in the cryptococcosis epidemiology as reservoir and carrier for C. neoformans var. neoformans, but also for other Cryptococcus species of increasing clinical interest.     

Genotyping of Turkish environmental Cryptococcus neoformans var. neoformans isolates by pulsed field gel electrophoresis and mating type

A total of 26 environmental Cryptococcus neoformans var. neoformans strains isolated from 634 samples of pigeon droppings collected from 54 different provinces of Turkey in 1996 and 1997 were included in this study. The results of pulsed-field gel electrophoresis (PFGE) showed that the 26 strains could be separated into 24 different PFGE patterns. In a mating-type study, of 26 strains, 20 were MATalpha, four were MATa, one was MATa/alpha and one was non-typable by STE20 specific primers. By the polymerase chain reaction typing, all the isolates were serotype A. The extensive heterogeneity among these isolates suggests that a single clonal population may not be present in Turkey. Additionally, the presence of an AMATa/DMATalpha hybrid may indicate the existence of strains that are AMATa mating type in Turkish environment.