Comparison of Different In Vitro Tests to Detect <Emphasis Type="Italic">Cryptococcus neoformans</Emphasis> Not Susceptible to Amphotericin B

Infections due to Cryptococcus neoformans cause severe disease, mostly in AIDS patients. The antifungal drug recommended for the initial treatment of these infections is amphotericin B with or without flucytosine, but treatment failure occurs, associated with high mortality. Thus, antifungal susceptibility testing is needed. However, the in vitro susceptibility tests available for C. neoformans are not useful to detect isolates that are not susceptible to antifungal agents such as amphotericin B. The aims of the present study were: (1) to determine and compare the in vitro activity of amphotericin B against C. neoformans clinical isolates by using different dilution and diffusion methods; (2) to evaluate the concordance among the methods used and the reference method; (3) to evaluate which method could be the best to correlate with the clinical outcome. The reference method EDef 7.2 from the European Committee on Antimicrobial Susceptibility Testing and commercial Etest strips were used to determine the minimal inhibitory concentration against amphotericin B. curves, minimal fungicidal concentration, and a disk diffusion method were also developed to evaluate the cidal activity of amphotericin B. The time–kill curve assay showed correlation (p < 0.05) with clinical outcome, whereas EDef 7.2, minimal fungicidal concentration, Etest, and disk diffusion showed no correlation (p > 0.05). Thus, the time–kill curve assay could be a potential tool to guide a more efficient treatment when amphotericin B is used.     

Minimal inhibitory concentrations of Candida species to five antifungals by using the reference dilution micromethod and the E-test.

It is accepted that the frequency of candidosis has increased during the last decade, specially in hospitalized patients. The more frequent use of azole antifungals and the recognition of isolates of Candida sp resistant to these and other drugs such as 5-fluorocytosine constitute a great need for a reproducible and useful C. albicans in vitro susceptibility testing method for monitoring antifungal therapy in clinical mycological laboratories. The E-test is a novel agar diffussion technique for testing the susceptibility of yeasts against a defined continous gradient of drug and could be used by most clinical laboratories. In this study the E-test and the NCCLS reference microbroth method (M27-P guidelines) were used to determine the MICs of amphotericin B, 5-flucytosine, itraconazole, fluconazole and ketoconazole for 50 clinical isolates of Candida albicans, Torulopsis glabrata, C. tropicalis and Hansenula anomala and five reference ATCC strains. The main purpose of the study was to compare the results obtained by the two methods. In general good agreement (+/- 1 dilution) was otained between both methods, despite differences observed for some species-antifungal combinations in which the MICs were lower by the E-test than by the microbroth method. MICs for C. albicans and T. glabrata to amphotericin B were < 0.50 microg/mL. Two isolates of C. albicans and two others of H. anomala, showed MIC < 8 microg/mL for 5- flucytosine. All isolates of T. glabrata and 40% of C. albicans showed MICs > 16 microg/mL for fluconazole. The results of this study indicate that E-test is an alternative for susceptibility testing to the NCCLS reference method. Because its simplicity it seems to be an easier test for routine clinical laboratories.     

In vitro antifungal resistance in Candida albicans from HIV-infected patients with and without oral candidosis.

The main purpose of this study has been to determine the in vitro antifungal susceptibility of clinical isolates from HIV-infected or AIDS patients, depending on the presence of oral candidosis. The oral cavity of 307 HIV-infected or AIDS patients was examined and an oral swab was cultured on Sabouraud glucose agar and studied by conventional mycological methods. In vitro antifungal susceptibility to amphotericin B, nystatin, fluconazole, itraconazole and ketoconazole was tested by disk diffusion with Neo-Sensitabs tablets (Rosco Diagnostica, Dinamarca). One hundred and thirty five Candida albicans isolates (91 serotype A, 38 serotype B, three C. albicans variety stellatoidea and three untyped isolates), three Candida krusei and two Candida glabrata were obtained. All the isolates were susceptible to nystatin and amphotericin B. However, 7.9% isolates were resistant to fluconazole and 2.9% isolates were resistant to ketoconazole or itraconazole. Nearly all C. krusei and C. glabrata isolates, 31% patients with candidosis and 20% Candida-colonized patients showed decreased susceptibility to azoles. This study shows that polyenes had a great in vitro efficacy against clinical isolates from HIV-infected patients and that in vitro resistance to azoles is not as high as observed in other countries.     

急性淋巴细胞白血病患者侵袭性头状地霉感染1例及文献回顾

目的:通过报道1例急性淋巴细胞白血病患者侵袭性头状地霉感染的临床资料,并结合文献探讨头状地霉感染的临床特点、有效的诊断及治疗方法。方法:报道国内首例急性淋巴细胞白血病患者化疗后骨髓抑制期感染头状地霉病例,并对该病的诊断及治疗等进行系统文献回顾。结果:该白血病患者经血培养证实为头状地霉感染,并累及肺脏、肝脏和皮肤,治疗过程中先后采用卡泊芬净、脂质体两性霉素B和脂质体两性霉素B联合伏立康唑等治疗,虽然脂质体两性霉素B联合伏立康唑治疗患者体温正常,临床症状稍有改善,但是患者在化疗后40天放弃治疗并死于心肺功能衰竭。结论:头状地霉感染的发病率低,临床症状不够典型,诊断困难,预后差。根据患者的临床表现,结合血培养、GM实验、G实验和CT扫描等检查,可有助于诊断。头状地霉感染尚无非常有效的治疗方式,采用脂质体两性霉素B或两性霉素B联合伏立康唑或其他新的抗真菌药物可能获得一定的疗效,早期诊断、早期联合治疗和患者早期脱离粒缺状态是治疗成功的关键。     

Comparative evaluation of standard dilution method and commercial kit for frozen plate antifungal susceptibility testing of yeasts using 200 clinical isolates

A comparative evaluation of standard microdilution methods and a commercial kit for frozen plate antifungal susceptibility testing of yeasts was performed using amphotericin B, flucytosine, fluconazole, miconazole, and itraconazole on 200 yeast isolates. The isolates included 100 strains of Candida albicans, eight of C. tropicalis, twelve of C. parapsilosis, eight of C. glabrata, five of Cryptococcus neoformans, thirteen of Trichosporon asahii, and 54 other strains of seven other species of ascomycotic yeasts. Microdilution testing was performed according to the standard method for antifungal susceptibility testing published by the Japanese Society for Medical Mycology (JSMM), which are a modification of the method developed by the National Committee for Clinical Laboratory Standards (NCCLS) M27-P. The commercial kit was prepared according to the manufacturer's instructions. The degree of agreement within +/-1 dilution for 200 clinical isolates against five antifungal agents was excellent with values for amphotericin B, flucytosine, fluconazole, miconazole, and itraconazole of 100%, 99.0%, 97.5%, 97.0%, and 97.0%, respectively. Overall, the frozen plate antifungal susceptibility testing kit provided convenient and reproducible results comparable to those obtained with the JSMM standard method.     

Use of spectrophotometric reading for in vitro antifungal susceptibility testing of Aspergillus spp

A comparative study of visual and spectrophotometric MIC endpoint determinations for antifungal susceptibility testing of Aspergillus species was performed. A broth microdilution method adapted from the National Committee for Clinical Laboratory Standards (NCCLS) was used for susceptibility testing of 180 clinical isolates of Aspergillus species against amphotericin B and itraconazole. MICs were determined visually and spectrophotometrically at 490 nm after 24, 48, and 72 h of incubation, and MIC pairs were compared. The agreement between the two methods was 99% for amphotericin B and ranged from 95 to 98% for itraconazole. It is concluded that spectrophotometric MIC endpoint determination is a valuable alternative to the visual reference method for susceptibility testing of Aspergillus species.     

<Emphasis Type="Italic">Acremonium strictum</Emphasis> Fungaemia in a Paediatric Immunocompromised Patient: Diagnosis and Treatment Difficulties

During the past two decades, an increasing number of unusual moulds has been reported as responsible for septicaemia and systemic or disseminated infections in immunocompromised patients. Investigation of fever in a 10-year-old boy with acute myeloblastic leukaemia, including blood cultures on selective media, allowed the diagnosis of a fungaemia due to the slow-growing fungus Acremonium strictum. The patient recovered with liposomal amphotericin B (AmB) and voriconazole, followed by voriconazole alone due to AmB resistance. Facing a neutropenic patient with fever, clinicians usually suspect bacterial or viral aetiologies. This case, however, illustrates the need for mycological analysis of blood samples in febrile neutropenic patients and for antifungal susceptibility testing.     

In Vitro Susceptibility of Environmental Isolates of Exophiala dermatitidis to Five Antifungal Drugs

Several dematiaceous fungi frequently isolated from nature are involved in cases of superficial lesions to lethal cerebral infections. Antifungal susceptibility data on environmental and clinical isolates are still sparse despite the advances in testing methods. The objective of this study was to examine the activities of 5-flucytosine, amphotericin B, itraconazole, voriconazole and terbinafine against environmental isolates of Exophiala strains by minimum inhibition concentration (MIC) determination. The strains were obtained from hydrocarbon-contaminated soil, ant cuticle and fungal pellets from the infrabuccal pocket of attine gynes. Broth microdilution assay using M38-A2 reference methodology for the five antifungal drugs and DNA sequencing for fungal identification were applied. Terbinafine was the most active drug against the tested strains. It was observed that amphotericin B was less effective, notably against Exophiala spinifera, also studied. High MICs of 5-flucytosine against Exophiala dermatitidis occurred. This finding highlights the relevance of studies on the antifungal resistance of these potential opportunistic species. Our results also contribute to a future improvement of the standard methods to access the drug efficacy currently applied to black fungi.     

Microbiological Characteristics and Susceptibility Patterns of Strains of Rhodotorula Isolated from Clinical Samples

The members of the genus Rhodotorula show a marked ubiquity. In man, they have been isolated from faeces, nails, skin, sputum, digestive tract and adenoids, forming part of the normal human flora, although in recent years cases have been reported of both local and systemic infection by this yeast. There are virtually no studies in the literature on the sensitivity of this genus to the antifungal agents in common clinical use. Therefore, it is considered of interest to study the microbiological characteristics and the susceptibility patterns of Rhodotorula isolated from clinical samples. A total of 35 different strains of Rhodotorula were studied. In vitro susceptibility testing to 5-fluorocytosine, amphotericin B, ketoconazole, fluconazole and itraconazole was performed. All the strains were considered sensitive to 5-fluorocytosine, amphotericin B, ketoconazole and itraconazole and resistant to fluconazole. As a conclusion, we can state that all the antifungal agents tested, except fluconazole, are useful medicaments for the treatment of infections by the Rhodotorula genus. This revised version was published online in June 2006 with corrections to the Cover Date.     

AmBisome,tres retos: infección por Candida auris,infección del sistema nervioso central e infección asociada a biopelículas

The treatment of invasive fungal infections remains a challenge, both for the diagnosis and for the need of providing the appropriate antifungal therapy. Candida auris is a pathogenic yeast that is responsible for hospital outbreaks, especially in intensive care units; it is characterized by a high resistance to the antifungal agents and can become multidrug-resistant. At present, the recommended antifungal agents for the invasive infections with this pathogen are echinocandins, always after carrying out an antifungal susceptibility testing. In case of no clinical response or persistent candidemia, the addition of liposomal amphotericin B or isavuconazole may be considered. Both fungal infection of the central nervous system and that associated with biomedical devices remain rare entities affecting mainly immunocompromised patients. However, an increase in their incidence in recent years, along with high morbidity and mortality, has been shown. The treatment of these infections is conditioned by the limited knowledge of the pharmacokinetic properties of antifungals. A better understanding of the pharmacokinetic and pharmacodynamic parameters of the different antifungals is essential to determine the efficacy of the antifungal agents in the treatment of these infections.     

In vitro susceptibility of dematiaceous fungi to ten antifungal drugs using an agar diffusion test

We assessed the usefulness of an agar diffusion method, NeoSensitabs, to determine in vitro sensitivity of 52 isolates of dematiaceous filamentous fungi against ten antifungal agents: amphotericin B, 5-fluorocytosine, ketoconazole, fluconazole, itraconazole, terbinafine, bifonazole, miconazole, clotrimazole, and griseofulvin. For the preparation of the inoculum, a spectrophotometric method including both Shadomy and Casitone agar (CAS) culture media was used. Dematiaceous filamentous fungi were sensitive to itraconazole, terbinafine and bifonazole. Ketoconazole (90.4%), miconazole (71%), and clotrimazole (46%) showed a variable susceptibility pattern. Most species were resistant to griseofulvin and fluconazole (96%). All isolates were resistant to 5-fluorocytosine. Sixty-three percent of strains were susceptible to amphotericin B and 28.8% resistant. Inhibition zones in the antifungal susceptibility testing did not vary according to culture medium, although fungal growth was better in CAS. Variations in antifungal sensitivity in Exophiala spinifera and Fonsecaea pedrosoi spp. would justify an in vitro susceptibility study when indicating antifungal therapy. These results show that NeoSensitabs agar diffusion method is simple, rapid, and low-cost and can be available to many clinical laboratories for the study of in vitro sensitivity of dematiceous moulds.     

阿萨希毛孢子菌生物膜构建及水杨酸干预策略研究

目的在医院内常用生物材料聚氯乙烯（PVC）表面构建阿萨希毛孢子菌的生物膜,评估该生物膜对几种临床抗真菌药物的耐药性,并观察水杨酸是否对阿萨希毛孢子菌生物膜的形成有干预作用。方法菌株鉴定采用API20CAUX并经PCR鉴定复核;使用PVC于RPM11640-MOPS中培养进行阿萨希毛孢子菌生物膜构建;MIC测定采用法国生物梅里埃公司ATB Fungus-3真菌药敏试剂条以及微量液体稀释法,并观察水杨酸对生物膜构建的影响。结果阿萨希毛孢子菌可以通过几个不连续阶段在聚氯乙烯表面形成生物膜,且已使用PVC块上附着的生物膜细胞比未使用PVC块上黏附的生物膜细胞明显密集;固着相即生物膜细胞的MIC比浮游相成倍提高;24h两性霉素B的MIC〉512μ/ml,且经两性霉素B的药物刺激后,阿萨希毛孢子明显可见芽管延长,菌丝交织;水杨酸作用后阿萨希毛孢子菌的菌丝明显变短,孢子短小。结论介入性器械可以作为阿萨希毛孢子菌生物膜构建的黏附基质,使微生物群体黏附于细胞外多聚材料表面而造成持续播散感染,因此生物膜干预对阿萨希毛孢子菌深部感染的治疗有很重要的意义。     

Disseminated Amphotericin-Resistant Fusariosis in Acute Leukemia Patients: Report of Two Cases

Disseminated fusariosis has emerged as a significant, usually fatal infection in immunocompromised hosts despite antifungal treatment. We describe here two patients with acute leukemia who developed disseminated amphotericin-resistant fusariosis, and review of six studies of cases series in the literature. Two Fusarium solani strains were isolated from blood and skin cultures of one patient, and one strain from the blood culture of the second patient. Both patients died despite antifungal treatment. Strains were identified by sequencing of ITS1 and ITS4 regions. Random amplified polymorphic DNA analysis of the three F. solani isolates showed a low degree of similarity. Screening for Fusarium spp. contaminants within our facility was negative. Using the CLSI M-38-A2 broth dilution method and E tests^®, we found that the MICs were low for voriconazole (0.12 and 0.5 mg/L, respectively), unexpectedly high for amphotericin B (≥8 and ≥32 μg/mL, respectively) and itraconazole (≥16 mg/ml). Patients with leukemia or persistent neutropenia should be assessed for disseminated fungal infections, including biopsy and skin cultures. Antifungal susceptibility tests are important due to the possibility of the strains being amphotericin resistant. Treatments must be aggressive, with high doses of antifungals or combined therapy.     

Subculture on potato dextrose agar as a complement to the broth microdilution assay for Malassezia pachydermatis

The main aim of this study was to verify the efficacy of subculture on potato dextrose agar (PDA) as a complement to the in vitro susceptibility test for Malassezia pachydermatis strains by a broth microdilution method, as well as to determine the MIC and MFC of azole derivatives, amphotericin B and caspofungin. The microdilution assay was performed in 96-well plates using a modified RPMI 1640 medium. The M. pachydermatis strains were resistant to caspofungin. All strains (n=50) had shown MIC values of <0.03, <0.03, 2.0, 4.0 and 4.0 mug/ml for itraconazole, ketoconazole, voriconazole, fluconazole and amphotericin B, respectively. Thus, the subculture on PDA improved the analysis of the in vitro antifungal susceptibility of M. pachydermatis.     

Azole Resistance in <Emphasis Type="Italic">Aspergillus fumigatus</Emphasis> in Patients with Cystic Fibrosis: A Matter of Concern?

Aspergillus fumigatus is the most frequent filamentous fungus isolated from respiratory specimens from patients with cystic fibrosis (CF). Triazoles are the most widely used antifungals in the treatment of allergic bronchopulmonary aspergillosis (ABPA) and invasive aspergillosis (IA) in CF patients. Treatment success could be severely compromised by the occurrence of azole-resistant A. fumigatus (ARAf), which is increasingly reported worldwide from both clinical samples and the environment. In previous studies, ARAf has been detected in up to 8% of CF patients. Isolates from CF patients requiring antifungal treatment should therefore be routinely subjected to antifungal susceptibility testing. The optimal treatment of ABPA or IA in CF patients with azole-resistant isolates has not been established; treatment options include liposomal amphotericin B i.v. and/or echinocandins i.v.     

Development of Reference Procedures for Broth Microdilution Antifungal Susceptibility Testing of Yeasts with Standardized Endpoint Determination

Standard guidelines for the broth microdilution antifungal susceptibility testing of amphotericin B, flucytosine, fluconazole, miconazole and itraconazole are reported. These are a modification of the method developed by the National Committee for Clinical Laboratory Standards (NCCLS) on the following two points: standardization of the means of endpoint determination and the inclusion of miconazole and itraconazole in the testing. MIC was determined to be when the positive control had a turbidity of 0.2 at the 630 nm wavelength. The endpoint was 80% inhibition for azoles and 100% inhibition for other drugs. The method provided good reproducibility, and a wide range of MIC distribution was observed in all antifungal agents except amphotericin B.     

Evaluation of <Emphasis Type="Italic">Fusarium solani</Emphasis> Hyphae and Conidia Susceptibility to Amphotericin B and Itraconazole: Study of a Clinical Case

Fusarium species are hyaline moulds belonging to the hyalohyphomycosis group that are usually found in the soil and plants. This organism has emerged as a cause of disseminated invasive disease. The correlation between in vitro value and clinical efficacy is low and many patients remain unresponsive to treatment despite in vitro susceptibility. We determined growth control for Fusarium solani using the BioCell-Tracer system that measures the growth rate of a single fungal hypha, and the effect of different concentrations of amphotericin B and itraconazole. The MIC for these two drugs was also determined by a broth microdilution technique, using RPMI 1640. Different MICs for amphotericin B were obtained by the two different methods. This paper describes a case of infection due to Fusarium solani in an allogeneic bone marrow transplanted patient, the microbiological diagnostic, antifungal susceptibility tests for conidia and hypha and clinical correlation.     

A Case Report of Penile Infection Caused by Fluconazole- and Terbinafine-Resistant <Emphasis Type="Italic">Candida albicans</Emphasis>

Candida albicans is the most common pathogen that causes balanoposthitis. It often causes recurrence of symptoms probably due to its antifungal resistance. A significant number of balanitis Candida albicans isolates are resistant to azole and terbinafine antifungal agents in vitro. However, balanoposthitis caused by fluconazole- and terbinafine-resistant Candida albicans has rarely been reported. Here, we describe a case of a recurrent penile infection caused by fluconazole- and terbinafine-resistant Candida albicans, as well as the treatments administered to this patient. The isolate from the patient was tested for drug susceptibility in vitro. It was sensitive to itraconazole, voriconazole, clotrimazole and amphotericin B, but not to terbinafine and fluconazole. Thus, oral itraconazole was administrated to this patient with resistant Candida albicans penile infection. The symptoms were improved, and mycological examination result was negative. Follow-up treatment of this patient for 3 months showed no recurrence.     

Should antifungal treatments be based upon results of antifungal susceptibility testing?

The development of reliable procedures for detecting resistance in vitro to antifungal agents and the increase in the prevalence of mycoses due to organisms with intrinsic or secondary resistance have reinforced the clinical relevance of antifungal susceptibility testing. A matter is currently in the limelight: can antifungal agents indications be based on susceptibility testing results? The present paper reviews the reliability of susceptibility testing procedures available at the moment, their capability for predicting therapeutic failure, and their clinical usefulness.