Cryptococcus laurentii fungemia in a premature neonate

Cryptococcus spp. other than Cryptococcus neoformans are generally considered nonpathogenic to humans. There are only 15 case reports of disease in humans caused by Cryptococcus laurentii infection. Underlying diseases and predisposing risk factors seem to play an important role in these cases. Our patient is the first case of an extremely low birth weight infant with C. laurentii fungemia reported in the English literature. In our case, the MIC of amphotericin B for C. laurentii was 0.25 to 1 microg/ml and the patient had a good outcome following the administration of amphotericin B at 10 mg/kg combined with central venous catheter removal. There will undoubtedly be an increasing occurrence of unusual fungal infections accompanying further advances in medicine. A high degree of suspicion and improvements in the techniques for culture and identification will contribute to the earlier diagnosis and treatment of unusual fungal infections.     

Sporothrix schenckii sensitivity to voriconazole, itraconazole and amphotericin B.

One hundred clinical isolates of Sporothrix schenckii were tested against voriconazole, itraconazole and amphotericin B using a modification of the NCCLS M27-A in vitro yeast susceptibility testing procedure. NCCLS M38-P for moulds was not used because yeast forms may have been present when the test isolates were incubated at 35 +/- 1 degrees C. The minimum inhibitory concentration (MIC) values were: voriconazole 0.5-8 (geometric mean titer 6.50) microg ml(-1) ; itraconazole 0.03-8 (geometric mean titer 1.56) microg ml(-1); and amphotericin B 0.25-2 (geometric mean titer 1.23) microg ml(-1). The minimum fungicidal concentration (MFC) values were: voriconazole 2-8 (geometric mean titer 7.67) microg ml(-1); itraconazole 0.125-8 (geometric mean titer 7.41) microg ml(-1); and amphotericin B 0.125-2 (geometric mean titer 1.53) microg ml(-1). Based upon MIC values, sensitivity to amphotericin B is strain-dependent. S. schenckii is more sensitive to itraconazole than voriconazole based upon a comparison of MIC geometric means, even though the MIC ranges were essentially the same.     

A flucytosine-resistant Cryptococcus neoformans (serotype D) strain isolated in turkey from cutaneous lesions.

A Cryptococcus neoformans strain from cutaneous lesions of a patient with thrombotic thrombocytopenia purpura was tested for in vitro susceptibility against seven conventional antifungal agents. The strain was susceptible to fluconazole, itraconazole, ketoconazole and miconazole but was resistant to 5-fluorocytosine (5-FC). Minimal inhibitory concentration (MIC) values obtained against amphotericin B and terbinafine were 1 and 4 microg ml(-1), respectively. The isolate belonged to serotype D. Few human cases of cryptococcosis have been reported over the last 50 years in Turkey. This is the first C. neoformans isolate in Turkey shown to have primary resistance to 5-FC. Primary resistance to flucytosine is rarely reported in C. neoformans and may be associated with treatment failure in some cases.     

In vitro antifungal susceptibility of Cryptococcus gattii

We have determined the in vitro susceptibilities of 57 strains of Cryptococcus gattii to nine antifungal agents and have compared the MICs for these strains with those for C. neoformans. MICs were determined by a microdilution reference method. Albaconazole and ravuconazole (MICs of 0.04 and 0.05 microg/ml, respectively) showed the best activities. Micafungin showed no activity (MIC of >128 microg/ml). In general, C. gattii was less susceptible than C. neoformans to all drugs tested, with the exception of amphotericin B and flucytosine.     

Global trends in the antifungal susceptibility of Cryptococcus neoformans (1990 to 2004)

The antifungal susceptibilities of 1,811 clinical isolates of Cryptococcus neoformans obtained from 100 laboratories in 5 geographic regions worldwide between 1990 and 2004 were determined. The MICs of amphotericin B, flucytosine, fluconazole, voriconazole, posaconazole, and ravuconazole were determined by the National Committee for Clinical Laboratory Standards broth microdilution method. Isolates were submitted to a central reference laboratory (University of Iowa) from study centers in Africa (5 centers, 395 isolates), Europe (14 centers, 102 isolates), Latin America (14 centers, 82 isolates), the Pacific region (7 centers, 50 isolates), and North America (60 centers, 1,182 isolates). Resistance to amphotericin B, flucytosine, and fluconazole was < or = 1% overall. Susceptibility to flucytosine (MIC, < or = 4 microg/ml) ranged from 35% in North America to 68% in Latin America. Similarly, only 75% of isolates from North America were susceptible to fluconazole (MIC, < or = 8 microg/ml) compared to 94 to 100% in the other regions. Isolates remained highly susceptible to amphotericin B (99% susceptibility at a MIC of < or = 1 microg/ml) over the entire 15-year period. Susceptibility to flucytosine (MIC, < or = 4 microg/ml) increased from 34% in 1990 to 1994 to 66% in 2000 to 2004. Susceptibility to fluconazole (MIC, < or = 8 microg/ml) increased from 72% in 1990 to 1994 to 96% in 2000 to 2004. Voriconazole, posaconazole, and ravuconazole all were very active (99% of isolates susceptible at MIC of < or = 1 microg/ml) against this geographically diverse collection of isolates. We conclude that in vitro resistance to antifungal agents used in the treatment of cryptococcosis remains uncommon among isolates of C. neoformans from five broad geographic regions and has not increased over a 15-year period.     

Interlaboratory evaluation of Etest method for testing antifungal susceptibilities of pathogenic yeasts to five antifungal agents by using Casitone agar and solidified RPMI 1640 medium with 2% glucose.

An interlaboratory evaluation (two centers) of the Etest method was conducted for testing the antifungal susceptibilities of yeasts. The MICs of amphotericin B, fluconazole, flucytosine, itraconazole, and ketoconazole were determined for 83 isolates of Candida spp., Cryptococcus neoformans, and Torulopsis glabrata. Two buffered (phosphate buffer) culture media were evaluated: solidified RPMI 1640 medium with 2% glucose and Casitone agar. MIC endpoints were determined after both 24 and 48 h of incubation at 35 degrees C. Analysis of 3,420 MICs demonstrated higher interlaboratory agreement (percentage of MIC pairs within a 2-dilution range) with Casitone medium than with RPMI 1640 medium when testing amphotericin B (84 to 90% versus 1 to 4%), itraconazole (87% versus 63 to 74%), and ketoconazole (94 to 96% versus 88 to 90%). In contrast, better interlaboratory reproducibility was determined between fluconazole MIC pairs when RPMI 1640 medium rather than Casitone medium was used (96 to 98% versus 77 to 90%). Comparison of the flucytosine MICs obtained with RPMI 1640 medium revealed greater than 80% reproducibility. The study suggests the potential value of the Etest as a convenient alternative method for testing the susceptibilities of yeasts. It also indicates the need for further optimization of medium formulations and MIC endpoint criteria to improve interlaboratory agreement.     

Timed-kill curves for Cryptococcus neoformans isolated from patients with AIDS.

Infection with Cryptococcus neoformans is an increasing problem in immunocompromised patients, particularly those with acquired immune deficiency syndrome (AIDS). Amphotericin B and fluconazole are currently acceptable therapies for cryptococcal meningitis; however, their effects remain suboptimal and recurrence or treatment failure is still a problem. Antifungal susceptibility testing may be an important tool for guiding therapy, but for C. neoformans, a reliable method is still not available. This retrospective study evaluated minimal inhibitory concentration (MIC) for amphotericin B and fluconazole, and minimal fungicidal concentration (MFC) and timed-kill curves for amphotericin B against 16 clinical isolates of C. neoformans obtained from AIDS patients with cryptococcal meningitis. No correlation between clinical outcome and MIC was observed for amphotericin B. In selected cases, the MFC seemed to be a better predictor of outcome than MIC. In this study, amphotericin B timed-kill curves appeared to show a correlation with clinical outcome of the 16 patients with AIDS-associated cryptococcal meningitis. These in vitro tests must be further evaluated in prospective studies to confirm their potential usefulness for guiding cryptococcal meningitis therapy.     

In Vitro Susceptibility of Environmental Cryptococcus neoformans Variety neoformans Isolates from Turkey to Six Antifungal Agents, Including SCH56592 and Voriconazole

 The in vitro antifungal susceptibility of 27 environmental (pigeon droppings) isolates of Cryptococcus neoformans var. neoformans, isolated from throughout Turkey, to six antifungal agents (amphotericin B, flucytosine, fluconazole, voriconazole, itraconazole, and SCH56592) was studied. Voriconazole, itraconazole, and SCH56592 all showed comparable activity and were more active than the remaining three antifungal agents tested. Overall, SCH56592 was the most active agent (MIC90, 0.015 μg/ml, at both 48 and 72 h), followed by itraconazole (MIC90, 0.03 μg/ml, at both 48 and 72 h) and voriconazole (MIC90, 0.25 μg/ml, at both 48 and 72 h), respectively. Antifungal susceptibility data for environmental isolates may reflect patterns for the clinical isolates recovered from patients from the same geographic area.     

Laccase and melanization in clinically important Cryptococcus species other than Cryptococcus neoformans

The laccase enzyme and melanin synthesis have been implicated as contributors to virulence in Cryptococcus neoformans. Since isolations of Cryptococcus species other than C. neoformans from clinical specimens have been increasing, we examined the laccase activities of C. albidus, C. laurentii, C. curvatus, and C. humicola. Incubation of cells with epinephrine produced adrenochrome color in C. albidus, C. laurentii, and C. curvatus but not in C. humicola. Activity was always less than in C. neoformans. Laccase was detected in the soluble fractions of disrupted C. albidus, C. laurentii, and C. curvatus cells. Activity staining of partially purified enzyme after nondenaturing polyacrylamide gel electrophoresis revealed that laccases from C. albidus, C. laurentii, and C. curvatus migrated more slowly than that from C. neoformans. One strain of C. curvatus exhibited two melanin bands. Thus, several clinically emerging Cryptococcus species express laccase and can synthesize melanin.     

In vitro susceptibilities and biotypes of Candida albicans isolates from the oral cavities of patients infected with human immunodeficiency virus.

Candida albicans strains were isolated from the oral cavities of 62 human immunodeficiency virus (HIV)-infected patients at different stages of HIV infection. Only patients with persistent generalized lymphadenopathy-acquired immunodeficiency syndrome (AIDS)-related complex or full-blown AIDS showed typical clinical symptoms for oral candidiasis. In general, the microbiological recovery of Candida strains from the oral cavity increased with more advanced stages of HIV infection. The antifungal activity of ketoconazole, itraconazole, nystatin, amphotericin B, and flucytosine against all 62 strains was evaluated by means of a photometer-read broth microdilution method for determination of the 30% inhibitory concentrations of the drugs. The 95% ranges of 30% inhibitory concentrations were as follows: less than or equal to 0.063 to 32 micrograms/ml for ketoconazole, less than or equal to 0.063 to 8 micrograms/ml for itraconazole, 0.5 to 4 micrograms/ml for nystatin, less than or equal to 0.063 to 4 micrograms/ml for amphotericin B, and less than or equal to 0.063 to 8 micrograms/ml for flucytosine. Two strains were resistant to flucytosine, one was resistant to ketoconazole, and three were resistant to itraconazole. Isolates from patients with full-blown AIDS showed significantly less susceptibility to itraconazole, amphotericin B, and flucytosine. Strains were biotyped by using the API 20C carbohydrate assimilation system. The major biotype accounted for 63.9% of the isolates. At repeated evaluation, a change in biotype pattern was seen in 27.3%.     

Extensive genetic diversity within the Dutch clinical Cryptococcus neoformans population

A set of 300 Dutch Cryptococcus neoformans isolates, obtained from 237 patients during 1977 to 2007, was investigated by determining the mating type, serotype, and AFLP and microsatellite genotype and susceptibility to seven antifungal compounds. Almost half of the studied cases were from HIV-infected patients, followed by a patient group of individuals with other underlying diseases and immunocompetent individuals. The majority of the isolates were mating type α and serotype A, followed by αD isolates and other minor categories. The most frequently observed genotype was AFLP1, distantly followed by AFLP2 and AFLP3. Microsatellite typing revealed a high genetic diversity among serotype A isolates but a lower diversity within the serotype D set of isolates. One patient was infected by multiple AFLP genotypes. Fluconazole and flucytosine had the highest geometric mean MICs of 2.9 and 3.5 μg/ml, respectively, while amphotericin B (0.24 μg/ml), itraconazole (0.08 μg/ml), voriconazole (0.07 μg/ml), posaconazole (0.06 μg/ml), and isavuconazole (0.03 μg/ml) had much lower geometric mean MICs. One isolate had a high flucytosine MIC (>64 μg/ml), while decreased susceptibility (≥16 μg/ml) for flucytosine and fluconazole was found in 9 and 10 C. neoformans isolates, respectively.     

Characterization of clinical strains of Aspergillus terreus complex: molecular identification and antifungal susceptibility to azoles and amphotericin B

We used molecular techniques to analyse 87 (n = 70 patients) Aspergillus terreus complex isolates, all of which were identified as A. terreus sensu stricto. The antifungal susceptibilities determined with CLSI M38-A2 (and Etest for amphotericin B) and expressed as mg/L for range of MIC/MIC(90) /geometric mean were as follows: itraconazole, 0.25-2/2/1.097; voriconazole, 0.125-2/2/1.176; posaconazole, 0.25-1/1/0.836; amphotericin B CLSI, 4-32/16/9.689; and Etest, 0.75-64/6/3.106. The MICs for amphotericin B were significantly higher than those found for the triazoles.     

Multisite reproducibility of the Etest MIC method for antifungal susceptibility testing of yeast isolates.

A multicenter study was performed to establish the interlaboratory reproducibility of Etest, to provide an additional comparison of Etest MICs with reference broth macrodilution MICs, and to develop some tentative quality control (QC) guidelines for using Etest for antifungal susceptibility testing of Candida spp. Two QC strains, Candida parapsilosis ATCC 22019 and Candida krusei ATCC 6258, were tested by Etest against amphotericin B, fluconazole, flucytosine, itraconazole, and ketoconazole in each of four laboratories. The QC strains were tested 20 times each against the five antifungal agents by using a common lot of RPMI agar. A total of 80 MICs per drug per strain were generated during the study. Overall, 98 to 100% of the MICs fell within a 3 log2 dilution range for the respective yeast-antifungal agent combinations. The level of agreement of Etest MICs with broth macrodilution MICs was 86 to 100% with amphotericin B (C. krusei and C. parapsilosis), itraconazole (C. krusei and C. parapsilosis), flucytosine (C. parapsilosis), and fluconazole (C. parapsilosis). A lower level of agreement was observed with ketoconazole (C. krusei and C. parapsilosis). Although all participants reported identical Etest MICs, the MICs of flucytosine and fluconazole when tested against C. krusei fell well above the upper limits of the reference range for this strain. The tentative QC limits for the two QC strains and five antifungal agents when tested by the Etest methodology are the same as the QC limits when tested by the reference broth macrodilution method for amphotericin B and C. krusei, itraconazole and C. krusei, flucytosine and C. parapsilosis, fluconazole and C. parapsilosis, and itraconazole and C. parapsilosis. The Etest QC ranges are 1 dilution broader (4-dilution range) than the reference macrodilution method QC ranges for ketoconazole and C. krusei, amphotericin B and C. parapsilosis, and ketoconazole and C. parapsilosis.     

Candida pseudorugosa sp. nov., a novel yeast species from sputum

Two yeast strains, strains XH 1026 and XH 1164, isolated from the sputum of an intensive care unit patient with acute pneumonia, were originally identified as Candida albicans and C. tropicalis, respectively. Sequence analysis of the 26S rRNA gene D1/D2 domain and the internal transcribed spacer (ITS) region indicated that the two strains represent a novel yeast species closely related to C. rogusa. The name Candida pseudorugosa sp. nov. is therefore proposed (type strain, AS 2.3107 [CBS 10433]). The new species is able to grow at 42 degrees C and is resistant or insusceptible to amphotericin B (MIC, 2 microg/ml), caspofungin (MIC, 64 microg/ml), itraconazole (MIC, 1 microg/ml), and nystatin (MIC, 16 microg/ml); dose-dependent susceptible to fluconazole (MIC, 16 microg/ml); and susceptible to flucytosine (MIC, 0.125 microg/ml) and voriconazole (MIC, 0.125 to 0.25 microg/ml). The code for C. pseudorugosa sp. nov. provided by the API 20C AUX system is identical to that for C. rugosa. The colonies of the new species on CHROMagar Candida appear blue-green, similar to those of C. albicans. In addition to the molecular method based on D1/D2 domain or ITS region sequencing, use of the combination of the API system and CHROMagar Candida is helpful for the correct identification of C. pseudorugosa sp. nov.     

Evaluation of Etest method for determining voriconazole and amphotericin B MICs for 162 clinical isolates of Cryptococcus neoformans

The performance of the Etest for voriconazole and amphotericin B susceptibility testing of 162 isolates of Cryptococcus neoformans was assessed against the National Committee for Clinical Laboratory Standards (NCCLS) broth microdilution method. The NCCLS method employed RPMI 1640 broth medium, and MICs were read after incubation for 72 h at 35 degrees C. MICs were determined by Etest for all 162 isolates with RPMI 1640 agar containing 2% glucose (RPG agar) and were read after incubation for 72 h at 35 degrees C. The Etest results for both voriconazole and amphotericin B correlated well with reference MICs. Agreement was 94% for voriconazole and 99% for amphotericin B. When discrepancy was noted between the results obtained by Etest and broth microdilution for voriconazole, the Etest generally provided a higher MIC. The Etest method using RPG agar appears to be a useful method for determining the susceptibility of C. neoformans to voriconazole and amphotericin B.     

Rapid presumptive identification of Cryptococcus neoformans by staphylococcal coagglutination.

A coagglutination reagent was prepared by sensitizing the Cowan I strain of Staphylococcus aureus with rabbit immune globulin directed against Cryptococcus neofromans A15 and absorbed with C. laurentii. This reagent was evaluated for its usefulness in differentiating C. neoformans from other yeast colonies rapidly. Antigen-containing extracts were prepared form Sabouraud dextrose agar cultures of 48 C. neoformans, 33 other Cryptococcus species, 21 Candida, 4 Torulopsis, 3 Saccharomyces, and 2 Rhodotorula strains. This was done by suspending a 0.001-ml loopful of colony growth in 0.5 ml of phenolized saline, mixing for 30 s, and then centrifuging. Equal volumes (50 microliters) of coagglutination reagent and yeast extract were mixed within marked circles on a glass slide and then mechanically rotated at 180 rpm for 8 min. Forty-five of the 48 strains of C. neoformans produced strong (3+ to 4+) agglutination, and 3 strains of serotype C produced weak (1+ to 2+) agglutination with the reagent. Other Cryptococcus species which reacted positively were 4 C. albidus subsp. diffluens, 7 C. albidus subsp. albidus, and 2 C. terreus strains; however, false-positive errors in identification were circumvented by performing a supplemental rapid test for nitrate utilization which differentiated these yeasts from C. neoformans. None of the other yeasts tested (including 14 C. laurentii, 2 C. luteolus, and 2 C. uniguttulatus strains) produced any degree of agglutination with the reagent. A commercial cryptococcal latex agglutination reagent (Crypto-Test, Microbiological Associates, Walkersville, Md.) proved less reliable for identifying C. neoformans yeast colonies because of cross-reactions which occurred with all other species of Cryptococcus tested.     

In vitro synergistic effect of minocycline combined with antifungals against Cryptococcus neoformans

BackgroundCryptococcus neoformans infections occur in immunocompromised patients, especially those with HIV infection, chemoradiotherapy after cancer, and organ transplantation. Infection can cause pneumonia and meningoencephalitis in severe cases with a high mortality rate if not treated. Although fluconazole and amphotericin B are the first-line treatments for cryptococcosis, the rate of fluconazole resistance has increased significantly due to long-term use. Minocycline is a derivative of tetracycline that exerts its antibacterial effect through inhibition of bacterial protein synthesis. It is also able to pass the blood-brain barrier to act on the central nervous system. The present study investigates the effects of minocycline in combination with antifungals in treating C. neoformans.ObjectiveTo determine in vitro interactions of minocycline combined with itraconazole, voriconazole, posaconazole, fluconazole and amphotericin B against C. neoformans.MethodsThe minimum inhibitory concentrations (MIC) of the antifungals were determined by the CLSI Clinical and Laboratory Standards Institute M27-A3 microdilution method. The in vitro synergistic effects of minocycline combined with itraconazole, voriconazole, posaconazole, fluconazole, and amphotericin B on C. neoformans were detected by the broth microdilution checkerboard technique and disk diffusion testing.Results and ConclusionThe working concentration ranges were 0.125–4 µg/mL for itraconazole, 0.03–0.125 µg/ml for voriconazole, 0.03–1 µg/ml for posaconazole, 0.25–16 µg/ml for fluconazole, and 0.125–2 µg/ml for amphotericin B. The synergistic rates of minocycline combinations against C. neoformans were 55% with itraconazole, 10% with voriconazole, 85% with posaconazole, 20% with fluconazole, and 70% with amphotericin B. The effective MIC value of minocycline in the synergistic combination decreased to 2–32 µg/ml, while the MIC of itraconazole decreased to 0.03–0.125 µg/ml, voriconazole 0.03–0.125 µg/ml, posaconazole 0.03–0.125 µg/ml, 0.125–4 µg/ml fluconazole, and 0.06–0.50 µg/ml amphotericin B. The disk diffusion assay showed that the plates containing minocycline and antifungal drugs produced inhibition zones with diameters larger than the single drug plates. Minocycline showed no antagonistic effect in the combinations. In conclusion, the combination of minocycline and azoles or amphotericin B has synergistic effects against C. neoformans in vitro.     

Antifungal susceptibility of Cryptococcus neoformans to amphotericin B and fluconazole

Cryptococcus neoformans has emerged as an important opportunistic fungal pathogen in immunocompromised individuals. The therapeutic options of C. neoformans an opportunistic fungal pathogen include flucytosine, amphotericin B, and azole agents. However in the present scenario, emergence of resistance has been reported, hence this study was undertaken to evaluate antifungal susceptibility pattern of C. neoformans isolates from this southern part of India. Ten isolates of C. neoformans were tested against Amp B and fluconazole, of which 7 were susceptible to both and a single isolate of C. neoformans var gatti was resistant to both with MIC of 32mg/ml and 64mg/ml respectively.     

Flucytosine Primary Resistance in Candida Species and Cryptococcus neoformans

 The in vitro activity of flucytosine (5FC) against 1,140 clinical isolates of Candida spp. and Cryptococcus neoformans was evaluated and compared with the activity of amphotericin B, fluconazole and itraconazole. Overall, 87.72% (1,000/1,140) of yeasts were susceptible to 5FC. This agent showed less potent in vitro activity against Candida glabrata, Candida krusei, Candida guilliermondii and Cryptococcus neoformans (MIC90s, 8–16 μg/ml) and intermediate activity or resistance to 6.5% of Candida albicans, 5.1% of Candida tropicalis and 0.8% of Candida parapsilosis strains. Amphotericin B showed potent activity against isolates with an MIC of 5FC≥8 μg/ml. A total of 112 of 140 strains that were 5FC-intermediate or -resistant showed decreased susceptibility to azoles (P<0.01).     

Fungemia in a cancer patient caused by fluconazole-resistant Cryptococcus laurentii.

We report the recent isolation of Cryptococcus laurentii from the blood of a patient given the diagnosis of ganglioneuroblastoma. The organism was identified using physiological and molecular characteristics, including morphology, carbohydrate and nitrate assimilation, urease activity, inability to form melanin on appropriate media, positive staining with diazonium blue B and sequence analysis of the D1/D2 domain of 26S ribosomal DNA. The isolate was resistant to fluconazole and 5-fluorocytosine using both the Etest and a broth microdilution assay. Repeated recovery of the organism from different blood cultures, and the patient's good response to treatment with amphotericin B support its etiological role. C. laurentii has rarely been implicated as a cause of clinically significant infections. The identity of reported isolates has not always been adequately documented, and some appear to have been isolated from lesions caused by Cryptococcus neoformans, emphasizing the true rarity of disease due to this fungus.