Clinical evaluation of a NASBA-based assay for detection of Candida spp. in blood and blood cultures

The number of life-threatening opportunistic fungal infections has shown a dramatic increase. However, the diagnosis of candidemia remains difficult. Nucleic acid amplification assays may improve the detection rate and decrease the time needed for detection and identification of Candida spp. Whole blood samples of patients suspected of having candidemia were analyzed using Nucleic Acid Sequence-Based Amplification (NASBA). Furthermore, aliquots of blood cultures of the patients after 2 days of culturing were tested. Eleven data sets from ten patients in two hospitals were generated. None of the whole blood samples was positive in the NASBA assay. Eight samples were positive in the NASBA assay after two days of culturing, whereas only two additional positive samples were found after longer incubation periods. Thus, a two-day culture step is sufficient to greatly improve the sensitivity of the NASBA assay. The NASBA assay detected Candida RNA in three patients. In one patient, the yeast was not detected by automated blood culturing, in another patient the NASBA assay detected the infection two days earlier than the blood culture system.     

Development and evaluation of the nuclisens basic kit NASBA for the detection of RNA from Candida species frequently resistant to antifungal drugs

We describe a Nucleic Acid Sequence Based Amplification (NASBA) protocol to detect 6 different Candida species (Candida krusei, Candida glabrata, Candida inconspicua, Candida dubliniensis, Candida norvegensis, Candida lusitaniae) and compare it to a PCR assay. NASBA showed a sensitivity of 1 Colony Forming Unit and detected RNA from all 6 Candida species within 1 working day. All 5 patients with documented candidiasis showed identical results by both methods. This assay offers a sensitive, specific and fast possibility to detect yeast RNA.     

基于M1磁珠富集血液中念珠菌快速检测方法的建立

  目的  建立3种基于重组人甘露聚糖结合凝集素（MBL）蛋白（M1蛋白）磁珠富集技术的血液微量念珠菌快速检测方法（荧光定量PCR法、微培养法、铺板培养法）。  方法  根据荧光定量PCR方法的扩增效果,比较3种裂解液（酵母裂解液、LSTE和TXTE）提取微量念珠菌DNA的效果;通过检测液体培养后的菌量,比较5种微培养液（PBS、SA、YPD、LB和BHI）的增菌效果;使用人血浆模拟样本评价建立的3种方法和标准血培养法对血液中微量念珠菌的检测效果。  结果  与TXTE和LSTE相比,酵母裂解液提取微量念珠菌DNA的效果最佳。 SA、YPD、LB和BHI 4种微培养液中的菌量随时间延长均有上升趋势,特别是BHI增菌效果最佳。 人血浆模拟样本中,荧光定量PCR法虽然能够在最短时间内（3.75 h）检测到病原菌,但不能检出全部阳性样本。 与传统血培养法相比,微培养法和铺板培养法可分别提前至少39 h和22 h获得检测结果,灵敏度为10菌落形成单位/mL（CFU/mL）。  结论  与传统血培养法相比,荧光定量PCR法、微培养法和铺板培养法均能有效缩短检测时间,并且有较高的特异性和灵敏度。     

Point prevalence,microbiology and fluconazole susceptibility patterns of yeast isolates colonizing the oral cavities of HIV-infected patients in the era of highly active antiretroviral therapy

We conducted a prospective study to address the prevalence and microbiological characteristics of yeast isolates colonizing the oral cavities of HIV-infected patients undergoing highly active antiretroviral therapy. Sixty-eight patients (67%) from a total of 102 were found to be colonized with yeasts. Sixty-five patients carried a single species (60 Candida albicans, three Candida glabrata and two Candida krusei) and three patients had mixed colonization of C. albicans and C. krusei. The status of yeast carrier was not associated with the number of CD4 cells or the viral load. Similarly, the type of antiretroviral regimen was not associated with the carriage of Candida spp. The only predictor of Candida colonization was a previous history of oropharyngeal candidiasis (P = 0.009). Although many patients in this series had already been treated with repeated courses of fluconazole therapy for previous episodes of oropharyngeal candidiasis, fluconazole susceptibility patterns showed that 93% of yeasts were susceptible to this triazole in vitro (MIC < or = 8.0 mg/L).     

Clinical comparison of the Bactec Mycosis IC/F, BacT/Alert FA, and BacT/Alert FN blood culture vials for the detection of candidemia

The present study analyzed the performance of Bactec Mycosis IC/F, BacT/Alert FA, and BacT/Alert FN vials in detection and time to detection (TTD) of Candida spp. in 179 simultaneous blood cultures. The Mycosis IC/F, BacT/Alert FA, and BacT/Alert FN vials could detect Candida spp. in 144 (80.45%) of 179, 149 (83.24%) of 179, and 8 (4.47%) of 179 samples, respectively. With the presence of antifungal therapy, the numbers of positive vials were higher in BacT/Alert FA compared to Mycosis IC/F, 87/99 versus 73/99, respectively (P < 0.05). TTD (SD) for C. albicans was shorter in Mycosis IC/F than in BacT/Alert FA vials without antifungal therapy, 20.89 (9.33) versus 28.26 (9.77), respectively (P < 0.01). The detection of Candida spp., with concomitant bacteremia, was higher in Mycosis IC/F than in BacT/Alert FA vials, 28/30 and 19/30, respectively (P = 0.01). The present data show that the use of Bactec Mycosis IC/F together with BacT/Alert FA vials might improve the detection of Candida spp.     

Longitudinal 10-year prospective survey of fungaemia in Slovak Republic: trends in etiology in 310 episodes. Slovak Fungaemia study group

A 10-year prospective survey of fungaemia in the Slovak Republic, involving 31 microbiology laboratories and 71 hospitals, was conducted from 1989-1998 (10 years): 310 fungaemias were analyzed for etiology, clinical characteristics, therapy, and outcome. C. albicans was responsible for 191 (61.6%) fungaemias, non-albicans Candida spp. (NAC) for 97 (31.3%), non-Candida yeasts for 18 (5.8%) and moulds (Fulsarium spp.) for four fungaemias. The most frequent NAC isolated from blood cultures were C. parapsilosis--30 (9.7%), C. krusei--18 (5.8%), C. tropicalis--14 (4.5%), and C. glabrata--10 (3.2%). Secular trends in etiology showed a sustaining decrease of C. albicans (from 100% in 1989 to 50.7% in 1998) and increase of NAC (from 0% in 1989-1990 to 46.3% in 1998). Non-Candida yeasts and moulds showed a stable proportion during the investigated period. There were statistically significant differences in etiology of fungaemia various subgroups of patients: non-albicans Candida spp. was significantly more frequent observed among subgroups of patients with pancreatitis and coma (53.3% vs. 31.3%, p < or = 0.02) and less frequently in the subgroup of neonates (15.0% vs. 31.3%, p < or = 0.006). Vice versa, C. albicans appeared more frequently in neonates (85%).     

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.     

Detection and identification of Candida spp. in human serum by LightCycler real-time polymerase chain reaction

The aim of this work was to develop LightCycler real-time polymerase chain reaction method to allow rapid detection and identification of Candida spp. in human serum with panfungal primers (internal transcribed spacer [ITS] and L18). Melting-curve analysis of the ITS sequences showed that each amplicon presented a specific melting point and enabled identification of 5 Candida spp. After parameters optimization, 58 sera were preliminary analyzed from 23 patients. For L18 primers, the LightCycler system enabled detection of DNA in 92% of patients with positive blood culture. These primers were not able to differentiate between species of Candida. By using ITS primers, the LightCycler system enabled detection of DNA in sera from 76.9% of patients with positive blood culture. With ITS primers, the species responsible for the infection was identified for 11 patients. These data revealed the LightCycler as a potential tool for early detection and identification of Candida.     

Antifungal susceptibility testing of yeasts: evaluation of technical variables for test automation.

The technical parameters for antifungal susceptibility testing with Candida species were reexamined to determine the optimal conditions for testing with semiautomated preparations of broth microdilution cultures, automated spectrophotometric readings of the cultures, and dose-response and endpoint determinations by means of a computer spreadsheet. Tests were based on proposed standard method M27P of the National Committee for Clinical Laboratory Standards for antifungal agents. RPMI 1640 broth with extra glucose to a final concentration of 2% gave higher and more reproducible drug-free control readings without affecting susceptibility endpoint readings. An inoculum of 8 x 10(4) yeasts per ml prepared from a carbon-limiting broth culture without further standardization was found to give optimal control readings after 48 h of incubation at 37 degrees C. For flucytosine, fluconazole, itraconazole, and ketoconazole, endpoints based on 50% growth inhibition (50% inhibitory concentration) gave the minimum variation with inoculum size and the fewest endpoint differences with RPMI 1640 medium obtained from two different suppliers. The 50% inhibitory concentration was also the optimal endpoint for fluconazole and ketoconazole susceptibilities in comparison with broth macrodilution MICs determined by the method of the National Committee for Clinical Laboratory Standards. Intralaboratory reproducibility was determined by retrospective analysis of replicate results for isolates retested at random over a 2-year period. This approach showed less favorable reproducibility than has been reported from purpose-designed, prospective antifungal susceptibility studies, but it may better reflect real-life test reproducibility. Susceptibility data for 616 clinical isolates of yeasts, representing 16 Candida and Saccharomyces spp., confirmed the tendency of Candida lusitaniae isolates to show relatively low susceptibilities to amphotericin B, the tendency of Candida krusei isolates to show low flucytosine and fluconazole susceptibilities, and the presence of some isolates in the species Candida albicans, Candida glabrata, and Candida tropicalis with low susceptibilities to azole derivative antifungal agents. The study demonstrates the value of automation and standardization in all stages of yeast susceptibility testing, from plate preparation to data analysis.     

泌尿系患者真菌感染的病原学和耐药性研究

目的研究住院患者泌尿系真菌感染病原学特点及其耐药性,为泌尿系真菌感染的防治提供参考数据。方法通过对尿液标本细菌学检验和分离鉴定以及药敏试验,对该医院临床科室泌尿系感染患者尿液标本进行了检测与分析。结果该医院2010年度从28 804例住院患者的尿液标本中检出真菌492株,真菌检出率为1.71%;其中从4 042例中段尿中检出真菌484株,检出率为11.97%。尿液标本中真菌检出率最高的患者来自重症医学科,其次是泌尿科和神经外科。尿液标本检出的真菌中,以热带假丝酵母菌居首位,构成比为74.4%;其次为白色假丝酵母菌和光滑假丝酵母菌。居前3位的真菌中,以光滑假丝酵母菌耐药性最强。结论泌尿系感染患者采集中段尿真菌检出率最高,以热带假丝酵母菌为主,主要患者分布在重症医学科,以光滑假丝酵母菌耐药性最强。     

A two year global evaluation of the susceptibility of Candida species to fluconazole by disk diffusion.

The in-vitro activity of fluconazole against 46,831 yeast isolates collected over a two-year period from 57 laboratories in 33 countries worldwide was assessed using a disc diffusion method. Candida albicans was the organism isolated most frequently, accounting for 68.6% of the total number of isolates. C. glabrata, C. tropicalis, C parapsilosis and C. krusei and Cryptococcus neoformans represented 9.9, 4.7, 4.3, 1.9, and 1.4% of isolates respectively during the 2 year period and rates varied markedly between countries. In 1999 data blood isolates represented 4.9% of all isolates and intensive care unit isolates represented 9.9%. In both the 1998 and 1999 data, 99% of C. albicans were fully susceptible (S) to fluconazole, and 95.6% of all species of yeasts tested were S or susceptible-dose dependent (S-DD) to fluconazole. No emerging trends of resistance were noted with any of the Candida spp. tested as 96% of all isolates retained susceptibility (S or S-DD) to this agent.     

Comparison of Albicans ID2 agar plate with the germ tube for presumptive identification of Candida albicans

Albicans ID2 (bioMérieux, France) is a commercially available chromogenic medium that allows rapid and specific macroscopic identification of Candida albicans and facilitates the differentiation of species in mixed cultures. We compared it with the standard method for the identification of yeast species, the germ tube test (GT). This study involved 423 clinical isolates, including 163 C. albicans and 260 non-albicans yeasts. Sensitivity of Albicans ID2 agar plates regarding the identification of C. albicans were 98.2% after 48 h of incubation and specificity of 96.6%. This method using rapid enzymatic method shows the same similar sensitivity than the GT test The false negative rate (1.8%) for the GT test is consistent with that previously reported. None tests discriminated between C. albicans and C. dubliniensis isolates.     

Fluconazole in the treatment of Candida-associated denture stomatitis.

A double-blind trial was carried out to study the effect of oral administration of fluconazole in the treatment of Candida-associated denture stomatitis. The study group consisted of 38 denture stomatitis patients who harbored yeasts, predominantly Candida spp., in significant numbers as determined by culture from the lesions. Half of the patients received 50 mg of fluconazole per day orally for 14 days, and the other half received placebo capsules. The following parameters were studied: degree of palatal erythema, presence of yeast cells (by plate count and microscopy of smears), identification to the species level of dominant yeast organisms, biotyping of Candida albicans, and treatment-related side effects. A significant reduction of erythema was seen after treatment with fluconazole, but the inflammation showed partial relapse 2 to 4 weeks after treatment was terminated. Reduced soreness of the oral mucosa was reported by six of the patients in the fluconazole group. No significant clinical or yeast flora changes were observed in the placebo group. Extensive changes in the yeast flora were observed in the fluconazole group, both in quantity and in composition of yeast species and C. albicans strains (biotypes), which perhaps indicated differences in pathogenicity and fluconazole susceptibility among various yeast species and C. albicans strains. Fluconazole did not produce any changes in the results of blood and urine analyses. The results indicate that fluconazole is a safe and well-tolerated antimycotic drug. The transient clinical and antimycotic effect may have been due in part to the possibility that therapeutic concentrations of the drug were not reached beneath the fitting denture surface and within the denture plaque.     

Activity of caspofungin and voriconazole against clinical isolates of Candida and other medically important yeasts by the CLSI M-44A disk diffusion method with Neo-Sensitabs tablets

In vitro activity of caspofungin and voriconazole against 184 clinical isolates of Candida and other medically important yeasts in comparison with that of fluconazole, ketoconazole, itraconazole and amphotericin B was determined by using a disk diffusion method (Neo-Sensitabs) standardized according to the recommendations of the CLSI documents M44-A and M44-S1 (same medium: Mueller-Hinton plus methylene blue; inoculum and minimal inhibitory concentration/zone breakpoints). Seventy-two percent of clinical isolates were susceptible to caspofungin, 23.6% showed an intermediate susceptibility (most of them were Candida parapsilosis) and 4.3% were resistant (values for Candida spp. were 71.2, 23.8 and 5%, respectively). For voriconazole, 96.7% of clinical isolates were susceptible and 3.3% were resistant (Candida spp.: 96 and 3.8%, respectively). Both caspofungin and voriconazole showed high activity against a wide variety of clinically important yeasts.     

Susceptibility patterns of Candida species recovered from Canadian intensive care units

OBJECTIVE: The objective of this study was to determine the speciation and susceptibility patterns of Candida species recovered from Canadian intensive care units (ICUs) during a 1-day point-prevalence study on fungal colonization/infection in Canadian ICUs. METHODS AND SETTING: Blood, urine, respiratory tract, rectal, and wound fungal cultures were performed for 357 patients present at any time during a single-day 24-hour period in 35 Canadian ICUs. Comparative in vitro activities of amphotericin B, fluconazole, itraconazole, voriconazole, posaconazole, micafungin, anidulafungin, and aminocandin were determined. RESULTS: Four hundred fifteen yeasts (409 Candida species and 6 non-Candida yeasts) were recovered. Almost 50% of the patients were found to have positive respiratory tract or rectal cultures. Candida albicans accounted for 72% of the Candida species isolated, followed by Candida glabrata (16%), Candida tropicalis (5%), Candida parapsilosis (3%), Candida krusei (2%), and other Candida species or nonspeciated isolates (2%). Minimum inhibitory concentrations (milligrams per liter) at which 90% of the strains were inhibited were 0.06 for micafungin as well as anidulafungin, 0.12 for voriconazole, 0.25 for itraconazole, posaconazole, as well as aminocandin, 1 for amphotericin B, and 4 for fluconazole. Only 4% of the isolates were resistant to fluconazole and/or itraconazole. CONCLUSIONS: Candida albicans is the predominant species colonizing Canadian ICU patients. Overall, the triazoles, both older and new compounds, and the echinocandins have excellent in vitro antifungal activities against Candida species recovered from Canadian ICUs patients.     

Antifungal Resistance to Fluconazole and Echinocandins Is Not Emerging in Yeast Isolates Causing Fungemia in a Spanish Tertiary Care Center

Accurate knowledge of fungemia epidemiology requires identification of strains to the molecular level. Various studies have shown that the rate of resistance to fluconazole ranges from 2.5% to 9% in Candida spp. isolated from blood samples. However, trends in antifungal resistance have received little attention and have been studied only using CLSI M27-A3 methodology. We assessed the fungemia epidemiology in a large tertiary care institution in Madrid, Spain, by identifying isolates to the molecular level and performing antifungal susceptibility testing according to the updated breakpoints of European Committee for Antimicrobial Susceptibility Testing (EUCAST) definitive document (EDef) 7.2. We studied 613 isolates causing 598 episodes of fungemia in 544 patients admitted to our hospital (January 2007 to December 2013). Strains were identified after amplification and sequencing of the ITS1-5.8S-ITS2 region and further tested for in vitro susceptibility to amphotericin B, fluconazole, posaconazole, voriconazole, micafungin, and anidulafungin. Resistance was defined using EUCAST species-specific breakpoints, and epidemiological cutoff values (ECOFFs) were applied as tentative breakpoints. Most episodes were caused by Candida albicans (46%), Candida parapsilosis (28.7%), Candida glabrata (9.8%), and Candida tropicalis (8%). Molecular identification enabled us to better detect cryptic species of Candida guilliermondii and C. parapsilosis complexes and episodes of polyfungal fungemia. The overall percentage of fluconazole-resistant isolates was 5%, although it was higher in C. glabrata (8.6%) and non-Candida yeast isolates (47.4%). The rate of resistance to echinocandins was 4.4% and was mainly due to the presence of intrinsically resistant non-Candida species. Resistance mainly affected non-Candida yeasts. The rate of resistance to fluconazole and echinocandins did not change considerably during the study period.     

In vitro activity of A-192411.29, a novel antifungal lipopeptide

A-192411.29 is a novel antifungal agent derived from the structural template of the natural product echinocandin. The in vitro activity of A-192411.29 against common pathogenic yeasts was assessed by National Committee for Clinical Laboratory Standards method M27-A. It demonstrated broad-spectrum, fungicidal activity and was active against the most clinically relevant yeasts, such as Candida albicans, Candida tropicalis, and Candida glabrata, as well as less commonly encountered Candida species; in general, its potency on a weight basis was comparable to that of amphotericin B. It maintained potent in vitro activity against Candida strains with reduced susceptibilities to fluconazole and amphotericin B. The in vitro activity of A-192411.29 against Cryptococcus neoformans was comparable to its activity against Candida spp. However, A-192411.29 did not demonstrate complete growth inhibition of Aspergillus fumigatus by the broth microdilution method used. A-192411.29 possesses an antifungal profile comparable to or better than those of fluconazole and amphotericin B against pathogenic yeasts, including strains resistant to fluconazole or amphotericin B, suggesting that it may be a therapeutically useful new antifungal drug.     

Pseudooutbreak of Candida versatilitis fungemia in a microbiology laboratory

Candida versatilitis was isolated from 10 blood cultures that had been supplemented with olive oil to promote the growth of Malassezia spp., and from the stock olive oil bottle in the laboratory. This unusual non-pathogenic yeast isolate was readily identified by DNA sequencing methodology. This report also points out that care must be taken to ensure the sterility of supplements added to blood culture media.     

Performance of a rapid yeast test in detecting Candida spp. in the vagina

We compared the performance of a rapid vaginal yeast assay (Savvycheck) with that of microscopic examination of a Gram-stained smear and culture of vaginal discharge in detecting Candida spp. Two hundred thirty-one women with vaginal symptoms were studied prospectively. Vaginal specimens obtained from all participants were studied by the Savvycheck rapid yeast test, microscopic evaluation of Gram-stained vaginal smears, and yeast culture. Savvycheck rapid yeast test was positive in 79% of women with positive cultures and in 3.6% of women with negative cultures. The Savvycheck test detected yeasts in 93% of subjects with positive Gram stain and in 5.5% of subjects with negative Gram stain. The Savvycheck rapid yeast test showed 93% sensitivity, 95% specificity, and a 97% negative predictive value compared with the Gram stain. It revealed 79% sensitivity, 96% specificity, and an 87% negative predictive value compared with culture. The Savvycheck rapid yeast test can be used in the busy office instead of microscopy as a point-of-care tool for diagnosing vulvovaginal candidiasis. It can also reduce the need for yeast cultures in patients with vaginitis.