Evaluation of CHROMagar Candida for presumptive identification of clinically important Candida species

CHROMagar Candida is a recently described and differential medium for the isolation and the presumptive identification of clinically important yeasts. We evaluated it with 262 yeast strains from clinical specimens, including 173 Candida albicans, 21 Candida tropicalis, 8 Candida krusei, 49 Candida glabrata, and 12 strains of other yeast species. Strains were presumptively identified on the basis of colony color and texture. These observations were compared with conventional identification results. Candida albicans was identified correctly in 170 (98%) of the 173 strains. A total of 46 of the 205 specimens that were plated on CHROMagar contained mixed cultures of yeast. Thirty-seven (80%) of these mixed cultures were not detected in the original specimens. CHROMagar Candida was useful for the rapid presumptive identification of Candida albicans and facilitated the recognition of mixed cultures. For other yeast species, it may provide additional information to laboratories that do not regularly perform identifications beyond the germ tube test.     

Multicenter prospective surveillance of oral Candida dubliniensis among adult Brazilian human immunodeficiency virus-positive and AIDS patients.

The incidence of C. dubliniensis in South America has not yet been determined. In the present study, oral swab samples were taken from 108 HIV-infected/AIDS individuals attending 6 separate Brazilian HIV-treatment centers to determine the incidence of C. dubliniensis in this population. Swabs were plated onto CHROMagar Candida medium and 155 isolates, presumptively identified as C. albicans or C. dubliniensis were further investigated. In a preliminary screen for C. dubliniensis, 13 of the 155 isolates showed no or poor growth at 42 degrees C, and all them were subjected to randomly amplified polymorphic DNA (RAPD) and polymerase chain reaction (PCR) analysis using C. dubliniensis-specific primers. We confirmed that 4 out of 13 isolates were C. dubliniensis, representing an incidence rate of 2.8% for the Brazilian HIV-infected population infected with yeasts exhibiting green colonies on CHROMagar Candida. This value is significantly lower than those reported in Ireland and the United States.     

"Room temperature" use of CHROMagar Candida

The color of colonies of 9 Candida species was examined on the chromogenic medium CHROMagar Candida incubated for 24-72 h at 25 degrees C, 30 degrees C or 37 degrees C. Colors and colony forms characteristic of C. albicans, C. dubliniensis, C. krusei and C. tropicalis were formed most rapidly and with the deepest hues at 37 degrees C. After 48 h incubation at 25 degrees C, 9 of 48 C. albicans isolates gave pink colonies instead of the green colonies characteristic for the species, and the blue-purple colony color characteristic of C. tropicalis isolates was not formed until 48 h at 25 degrees C. Incubation of the chromogenic medium at temperatures below 30 degrees C cannot be recommended for reliable presumptive identification of Candida spp., and pink colonies of C. glabrata would not be reliably distinguished from pink colonies formed by other species under any of the incubation conditions used.     

Evaluation of Bichro-Dubli Fumouze to distinguish Candida dubliniensis from Candida albicans

We have evaluated the ability of the Bichro-Dubli Fumouze (Fumouze Diagnostics, Levallois-Perret, France) latex agglutination test to identify colonies of Candida dubliniensis grown on different media. The test was positive for 103 of 106 isolates of C. dubliniensis and negative for Candida albicans and other Candida species studied. The sensitivity and specificity of the test were 97.1% and 100%, respectively. The test is very rapid, simple, and reliable giving the same results independently of whether the colonies are grown previously on Sabouraud dextrose agar, CHROMagar Candida medium, Candida ID2 medium, or CHROMagar-Pal's medium.     

医院内假丝酵母菌感染的菌种构成和耐药性分析

目的 分析医院内感染假丝酵母菌菌种构成和耐药性。 方法 对临床标本分离的酵母样真菌,用VITEK-Ⅱ(生物梅里埃公司)生化鉴定仪、API20c生化鉴定试纸条和念珠菌显色琼脂以及聚合酶链反应(polymerase chain reaction, PCR)方法进行酵母样真菌菌种的鉴定,应用ATB Fungus3 进行药敏试验。 结果 96株酵母样真菌,可分为6个种,包括白色念珠菌40株 (41.7%)、热带念珠菌36株 (37.5%)、光滑念珠菌13株 (13.54%)、近平滑念珠菌5株 (5.21%)、克柔念珠菌1株 (1.04%)、挪威念珠菌 1株(1.04%)。各种假丝酵母菌对5种抗真菌药呈现不同的敏感性,对两性霉素B和5-氟胞嘧啶的敏感率为100%,而对氟康唑、伊曲康唑、伏立康唑则表现出一定的耐药性。 结论 基因间隔转录区分子生物学分析结合传统培养和生化方法,可有效提高假丝酵母菌鉴定的准确性。本研究结果提示医院内非白假丝酵母菌感染有增多趋势。重症监护病房(intensive care unit, ICU)是重要的假丝酵母菌来源科室。60岁以上的老龄患者是医院内真菌感染的高危人群。体外药敏试验提示部分假丝酵母菌出现了唑类药物(氟康唑和伊曲康唑)的耐药性。     

Evaluation of growth characteristics on blood agar and eosin methylene blue agar for the identification of Candida (Torulopsis) glabrata

Candida albicans and Candida (Torulopsis) glabrata are the most common species of yeast encountered in the clinical laboratory. In this study, we sought to evaluate simple means of screening cultures for the presence or absence of C. glabrata. Twelve thousand five hundred (12,500) consecutive cultures were evaluated for sufficient yeast growth to warrant identification. When detected (369 isolates), the amount of growth on eosin methylene blue agar (EMB) versus sheep blood agar (BAP) (both incubated in 5% CO₂), wet mount morphology, and germ tube production were evaluated. All germ tube-negative yeasts were definitively identified using the Vitek YBC card. Of the 369 yeast isolates included in this study, 255 were C. albicans, 102 C. glabrata, and 42 other Candida species. Growth on EMB was greater than BAP for 92 isolates; all identified as C. glabrata. When EMB growth was equal to or less than BAP, 10 isolates were C. glabrata and 267 were other Candida ssp. An accurate presumptive identification of C. glabrata may be made using the observation of greater growth on EMB versus BAP. When coupled with the germ tube test, the majority of yeast isolates could be identified by these simple methods in our laboratory.     

In Vitro Susceptibilities of Candida and Cryptococcus neoformans Isolates from Blood Cultures of Neutropenic Patients

Fluconazole-resistant Candida albicans and intrinsically fluconazole-resistant Candida species have been reported as bloodstream isolates. However, an association between the isolation of fluconazole-resistant Candida from the bloodstream and patient risk factors for fungemia has not been established. The purpose of this study was to determine the prevalence of fluconazole resistance in bloodstream isolates of Candida species and Cryptococcus neoformans collected from patients with neutropenia, one of the most important risk factors for fungemia. MICs of voriconazole, fluconazole, itraconazole, ketoconazole, amphotericin B, and flucytosine were determined by the National Committee for Clinical Laboratory Standards M27-A method (1997). Voriconazole, on a per-weight basis, was the most active azole tested. Fluconazole resistance (MIC >/= 64 microg/ml) was not identified in any of the C. albicans (n = 513), Candida parapsilosis (n = 78), Candida tropicalis (n = 62), or C. neoformans (n = 38) isolates tested.     

PCR-RFLP在VVC相关念珠菌菌种鉴定中的应用

目的:用限制性片段长度多态性聚合酶链反应(polymerase chain reaction-restriction fragment length polymorphism,PCR-RFLP)技术快速准确鉴定外阴阴道念珠菌病(VVC)相关念珠菌菌种,并对科玛嘉显色培养法、Vitek 2 YST鉴定卡和PCR-RFLP 3种方法鉴定念珠菌菌种的效果进行方法学评价。方法:收集贵阳市妇幼保健院VVC患者感染的念珠菌菌种100株,用十六烷基三甲基溴化铵法(cetyltrimethylammonium bromide,CTAB)法提取念珠菌DNA,PCR扩增念珠菌DNA的ITS片段并进行测序分析确定念珠菌菌种;分别采用科玛嘉显色培养法、Vitek 2 YST鉴定卡和PCR-RFLP 3种方法对其进行鉴定,以测序结果为“金标准”,比较3种方法鉴定念珠菌菌种的正确率。结果:科玛嘉显色培养法中5株葡萄牙念珠菌与2株酿酒念珠菌显色错误,均显淡紫色;Vitek 2 YST鉴定卡中,1株白念珠菌与2株热带念珠菌鉴定不出,Cyberlindnera fabianii、Candida orthopsilosis与Candida metapsilosis鉴定错误;PCR-RFLP采用内切酶MspⅠ可成功鉴定念珠菌中除Candida metapsilosis、Candida orthopsilosis与近平滑念珠菌之外的其他菌种,进一步采用内切酶ApaⅠ与NcoⅠ可将Candida metapsilosis、Candida Orthopsilosis与近平滑念珠菌三者鉴别开。科玛嘉显色培养法对白念珠菌、克柔念珠菌、热带念珠菌等3种念珠菌鉴定正确率100%,对光滑念珠菌的鉴定正确率为73.1%;Vitek 2 YST鉴定卡可鉴定常见念珠菌且正确率较高,不能鉴定Cyberlindnera fabianii、Candida orthopsilosis与Candida metapsilosis等非常见念珠菌;PCR-RFLP技术可快速准确鉴定所有念珠菌菌种。结论:PCR-RFLP技术为早期准确鉴定临床念珠菌感染提供了新的选择,具有很好的应用前景。     

Molecular characterization of Italian Candida parapsilosis isolates reveals the cryptic presence of the newly described species Candida orthopsilosis in blood cultures from newborns

The authors report the molecular characterization of Candida parapsilosis isolates recovered from the blood and venous central catheter tips of patients admitted to different care units of the Polyclinic Hospital, University of Messina, Italy. Among 97 presumed C. parapsilosis isolates examined, 94 were identified as C. parapsilosis sensu stricto and the remaining 3 isolates were found to belong to the cryptic species Candida orthopsilosis which was recovered only from blood cultures of neonates (<30 days old) born prematurely. No C. metapsilosis was found in this study. This study emphasizes the role of C. parapsilosis as an important nosocomial pathogen, and it also describes, for the first time, the occurrence of C. orthopsilosis in newborns.     

Susceptibility of nosocomial isolates of Candida species to LY121019 and other antifungal agents

LY121019 is a novel analog of the polypeptide antifungal antibiotic echinocandin B. We investigated the in vitro activity of LY121019, amphotericin B, ketoconazole and 5-fluorocytosine against 131 nosocomial isolates of Candida species: C. albicans (n = 50), C. tropicalis (n = 30), C. rugosa (n = 12), C. parapsilosis (n = 11), C. lusitaniae (n = 10), C. guillermondii (n = 9), and C. krusei (n = 9). In vitro susceptibility testing was performed using a broth microdilution method. The minimal inhibitory concentrations (MIC) of LY121019 were less than or equal to that of the other antifungal agents against C. albicans and C. tropicalis but were generally higher for the other species of Candida. Paradoxical growth at high concentrations, but not at low concentrations, of LY121019 was observed with isolates of C. albicans and C. tropicalis.     

Fluconazole susceptibilities of Candida species and distribution of species recovered from blood cultures over a 5-year period.

The distribution and fluconazole susceptibilities of Candida species isolated over a 5-year period were investigated. Susceptibilities were determined by using a new microtiter procedure and the National Committee for Clinical Laboratory Standards (NCCLS) proposed standard. The new method correlated well with the NCCLS proposed standard and gave very clear end points. Results indicate that there are species-related differences in MICs as reflected in the MICs for 90% of species tested. Candida albicans is most susceptible to fluconazole, while Candida glabrata is among the least susceptible. These findings coincided with the observation of a shift in distribution of yeast species recovered from blood cultures from 1987 to 1992. C. albicans was the predominant species (87%) in the pre- or early fluconazole years but decreased to only 31% of the isolates in 1992. Thus, Candida species for which MICs of fluconazole were higher have become more prominent in recent years. Significantly, throughout this period, MICs for each species did not change appreciably.     

中国侵袭性真菌耐药监测网成员单位重症监护室侵袭性酵母的分布特征及其对唑类药物敏感性的变迁

目的回顾性分析2010-2014年中国侵袭性真菌耐药监测网(CHIF-NET)62所监测中心重症监护室(ICU)侵袭性酵母的现状,了解我国侵袭性酵母分布的特征及其对唑类药物的耐药情况。方法收集各所监测中心初步鉴定的菌株,送至北京协和医院检验科,采用基质辅助激光解吸电离飞行时间质谱技术结合分子测序技术对所有菌株进行复核鉴定,再进行唑类药物敏感性检测。结果2010-2014年,62所监测中心ICU共检出侵袭性酵母2863株,其中以念珠菌属最多,计2771株。标本类型包括血液标本(50.8%,1453/2863),无菌体液标本(49.2%,1410/2863);其中,50.7%(1404/2771)的念珠菌属分离自血液标本。5年间,白念珠菌对氟康唑和伏立康唑十分敏感,敏感率>99.0%;热带念珠菌对氟康唑和伏立康唑的耐药率显著升高,均从12.2%升至23.1%(P<0.01);光滑念珠菌对氟康唑的耐药率也显著升高,从14.7%升至27.7%(P<0.01)。非念珠菌属中,新型隐球菌复合体对伏立康唑全部敏感,从2013年开始出现对氟康唑耐药菌;其他酵母对两种唑类药物耐药率整体较高,分别为43.6%和32.7%。菌株复核鉴定的正确率为86.2%(2467/2863)。结论ICU中侵袭性酵母分离株中以念珠菌属为主,主要分离自血液,氟康唑和伏立康唑对其抗菌作用非常显著;而非白念珠菌的耐药率有不同程度的升高及交叉耐药的出现,临床需要加以重视。     

Prospective Study of Candida Species in Patients at a Comprehensive Cancer Center

Since most nosocomial systemic yeast infections arise from the endogenous flora of the patient, we prospectively evaluated the species stratification and antifungal susceptibility profile of Candida spp. associated with heavy colonization and systemic infection in patients at Memorial Sloan-Kettering Cancer Center in New York. A total of 349 Candida isolates were obtained from 223 patients during the later half of 1998. Cancer was the most common underlying disease, occurring in 91% of the patients, including 61.8% with organ and 23.7% with hematological malignancies; 4.4% of the patients had AIDS. Candida albicans was the predominant species (67.3%); among 114 non-albicans Candida spp., C. glabrata (45.6%) was the most frequent, followed by C. tropicalis (18.4%), C. parapsilosis (16.6%), and C. krusei (9.6%). The overall resistance to triazole-based agents among all yeast isolates was 9.4 and 10.8% for fluconazole and itraconazole, respectively. A total of 5% of C. albicans strains were resistant to triazole antifungals, whereas 30.8 and 46.2% of C. glabrata strains were resistant to fluconazole (MIC > or = 64 microg/ml) and itraconazole (MIC > or = 1 microg/ml), respectively. A significant association was observed between prior treatment with triazole and isolation of fluconazole-resistant C. albicans (P = 0.005, OR 36), although this relationship was not seen in C. glabrata isolates (P = 0.4). This study reinforces the importance of periodic, prospective surveillance of clinical fungal isolates to determine appropriate prophylactic, empiric, and preemptive antifungal therapy for the highly susceptible patient population.     

影响念珠菌检验结果因素的探讨

目的探讨常规微生物学检验方法中影响念珠菌检验结果准确性的因素。方法用常规方法分离、培养、鉴定14株念珠菌并对其中10株作DNA序列分析,确定分子型别。结果不同品牌的念珠菌显色平板初步鉴定结果不同,科玛嘉显色平板只能对白念珠菌作初步鉴定;用ATB仪器鉴定时,为了得到准确的鉴定结果最好将板条放30℃孵育48h,DADE 4h快速酵母菌鉴定系统对临床常见酵母菌的鉴定结果与ATB一致;10株念珠菌分子分型结果中只有1株不支持自动化仪器鉴定结果。结论培养基、培养温度和时间都是影响念珠菌检验结果准确性的因素。     

Distribution and antifungal susceptibility of Candida species causing candidemia from 1996 to 1999

Susceptibilities to amphotericin B and fluconazole of 383 Candida species isolated from blood were determined. Candida albicans was the most common species (55.6%), followed by Candida parapsilosis (17.5%), Candida tropicalis (16.5%), Candida glabrata (5.2%), Candida guilliermondii (2.3%), and others (2.9%). All but three isolates, Candida ciferrii, C. tropicalis, and C. glabrata, one each, were susceptible to amphotericin B. A total of 367 (95.8%) and 15 (4.2%) isolates were susceptible and susceptible-dose dependent to fluconazole, respectively. Only one isolate, a C. glabrata, was resistant to fluconazole. Few patients (13%) having prior fluconazole treatments may explain the low rate of resistance to fluconazole in this study.     

Effects of caspofungin against Candida guilliermondii and Candida parapsilosis

The in vitro activity of caspofungin (CAS) was investigated against 28 yeast isolates belonging to Candida albicans (n = 5), Candida guilliermondii (n = 10), and Candida parapsilosis (n = 13). CAS MICs obtained by broth dilution and Etest methods clearly showed a rank order of susceptibility to the echinocandin compound with C. albicans > C. parapsilosis > C. guilliermondii. Similarly, time-kill assays performed on selected isolates showed that CAS was fungistatic against C. albicans and C. parapsilosis, while it did not exert any activity against C. guilliermondii. In a murine model of systemic candidiasis, CAS given at doses as low as 1 mg/kg of body weight/day was effective at reducing the kidney burden of mice infected with either C. albicans or C. guilliermondii isolates. Depending on the isolate tested, mice infected with C. parapsilosis responded to CAS given at 1 and/or 5 mg/kg/day. However, the overall CFU reduction for C. guilliermondii and C. parapsilosis was approximately 100-fold less than that for C. albicans. Our study shows that CAS was active in experimental systemic candidiasis due to C. guilliermondii and C. parapsilosis, but this activity required relatively high drug dosages.     

Evaluation of the MALDI TOF-MS method for identification of Candida strains isolated from blood cultures

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI TOF-MS) allows rapid and accurate identification of microorganisms. It is being used increasingly and becoming an important tool in clinical laboratories. Phenotypic identification of Candida species remains labor- and time consuming, and the results may sometimes be inconclusive. Rapid and reliable species identification of Candida is essential for antifungal treatment due to species-specific susceptibility patterns. In this study, we evaluated the performance of MALDI TOF-MS for identification of Candida strains. A total of 281 clinical Candida strains isolated from blood cultures were included in this study. Candida species were identified with conventional methods and automated VITEK 2 YST panels as well as with MALDI TOF-MS. Isolates with discrepant results were subjected to DNA sequencing of the internal transcribed spacer (ITS) region (ITS1-5.8S-ITS2). Ninety-four percent of the isolates were identified correctly by VITEK 2 and MALDI TOF-MS. Altogether, MALDI-TOF MS yielded the correct species identification for 281 (100%) clinical Candida isolates. MALDI-TOF proved to be a rapid and reliable method for identification of Candida strains in the clinical laboratory.     

Exposure to therapeutic concentrations of ritonavir, but not saquinavir, reduces secreted aspartyl proteinase of Candida parapsilosis

The effect of ritonavir and saquinavir, HIV proteinase inhibitors, on the secreted aspartyl proteinase (Sap) activity of Candida parapsilosis was studied. In a proteinase-inducing medium (yeast carbon base-bovine serum albumin), Sap activity in all clinical isolates of C. parapsilosis (n = 20) was observed at 37 degrees C but not at 22 degrees C. The presence of ritonavir at a concentration of 8 microg/ml produced an inhibition close to 50% albumin consumption and also delayed yeast growth; however, saquinavir did not have any effect on growth or on Sap activity. In Sabouraud broth, which does not induce Sap production, no effect was shown on yeast growth by either of the two HIV proteinase inhibitors studied.     

The use of Niger seed agar to screen for Candida dubliniensis in the clinical microbiology laboratory

Candida dubliniensis is a recently described pathogenic yeast that is closely related to C. albicans. The germ tube test is used routinely in diagnostic laboratories for the identification of C. albicans, and C. dubliniensis may also produce germ tubes under the same conditions. We evaluated a previously described method for differentiating between the two species using Niger seed agar (Staib agar). The aim was to find a useful, user-friendly and cost-effective method for use in diagnostic work. C. albicans produces only yeast cells on this medium after 24 h at 37 degrees C, while C. dubliniensis produces extensive hyphal and pseudohyphal growth that is easily observed. Of 495 yeasts isolated in, or sent for identification to, a diagnostic mycology laboratory 9 isolates (1.8%) were found to be C. dubliniensis. The method was found to be valuable for screening yeasts before proceeding to further identification if positive for hyphal/pseudohyphal growth on Niger seed agar. This method is therefore suitable for the screening of selected yeast isolates in order to identity C. dubliniensis and will further our understanding of the clinical importance of this species.     

The first Korean case of candidemia due to Candida dubliniensis

Candidemia due to uncommon Candida spp. appears to be increasing in incidence. C. dubliniensis has been increasingly recovered from individuals not infected with HIV. Identification of C. dubliniensis can be problematic in routine clinical practice due to its phenotypic resemblance to C. albicans. We report the first case of C. dubliniensis candidemia in Korea, which occurred in a 64-yr-old woman who presented with partial seizure, drowsiness, and recurrent fever. Germ-tube positive yeast that was isolated from blood and central venous catheter tip cultures formed smooth, white colonies on sheep blood agar and Sabouraud agar plates, indicative of Candida spp. C. dubliniensis was identified using the Vitek 2 system (bioMerieux, USA), latex agglutination, chromogenic agar, and multiplex PCR. The blood isolate was susceptible to flucytosine, fluconazole, voriconazole, and amphotericin B. After removal of the central venous catheter and initiation of fluconazole treatment, the patient's condition gradually improved, and she was cleared for discharge from our hospital. Both clinicians and microbiologists should be aware of predisposing factors to C. dubliniensis candidemia in order to promote early diagnosis and appropriate treatment.