Candidiasis is the most common fungal infection of blood [1, 2]. Candidemia in hospitalized patients is associated with a higher attributable mortality, ranging from 5%–71%, a significantly increased cost of healthcare, and an increased length of hospital stay [3, 4]. Despite advances in healthcare, invasive candidiasis-associated mortality has remained stable for the past 2 decades, although a decreased mortality rate has been observed for other fungal infections e.g. invasive aspergillosis . Candida albicans is the predominant Candida species accounting for over half of all cases of candidemia, but the incidence ofnon-albicans Candida species has been increasing in the last decade throughout the world. In particular, C. glabrata, C. tropicalis, and C. parapsilosis, now comprise infections in one-fourth of all patients with candidemia .
Fluconazole remains the initial choice of therapy for candidemia as recommended by the Infectious Disease Society of America . However, fluconazole-resistant strains, particularly of non-Candida albicans species, have been increasing in prevalence . The continuously changing patterns of Candida species and the rising incidence of antifungal-resistance in less-common species, which vary between different geographic areas, means that performing antifungal susceptability testing is crucial in guiding treatment strategies .
The broth microdilution (BMD) method is the standard approved reference method for Candida susceptibility testing according to Clinical and Laboratory Standard Institute (CLSI) guidelines . However, its complexity, requiring experienced laboratory technicians, limit its availability for routine laboratory testing in resource-limited hospitals. To overcome these limitations, agar-based methods including the E-test (ET) and disk diffusion (DD) methods have become attractive alternatives because of their simplicity, reproducibility, and lack of the need for specialized equipment. Other studies have suggested that ET and DD show comparable results to the standard method of BMD [10-12]. The DD method in particular, being widely commercially available and inexpensive (its cost is ten-fold lower than that of ET), has become the most acceptable alternative for community settings, although one study has reported low correlation with BMD for antifungal-resistant isolates .
The aim of the present study was to evaluate the performance of DD and ET assays compared with the standard BMD method, and to validate the accuracy of these agar-based methods for initial routine laboratory screening for antifungal susceptibilities of local strains in Thailand.
Materials and methods
Study design and data collection
A total of 63 isolates of Candida species were obtained routinely from the blood of patients with candidemia in King Chulalongkorn Memorial Hospital, a 1500-bed single tertiary-care center in Thailand, over a period of 3 years (January 2007 to December 2010), and identified in the Department of Microbiology. Isolates from same patient that were obtained within one week were considered as duplicates and excluded. BD BACTEC Myco/F Lytic culture medium (Becton, Dickinson and Company, Sparks, MD, USA) was used for culture of all Candida isolates included in this study. Coded (linked anonymized) isolates that could not be directly identified with patients were stored at -80°C in Sabouraud dextrose broth with beads (BD Diagnostics, Franklin lakes, NJ, USA). These isolates were then subcultured at least twice on Sabouraud dextrose agar at 35°C to confirm viability and purity before susceptibility testing in the present study. The present study was approved by the Institutional Review Board of the Faculty of Medicine, Chulalongkorn University (IRB 168/54 and 315/58, COA Nos. 258/ 2011 and 569/2015).
All isolates were cultured on chromogenic agar (CHROMagar, Paris, France) for presumptive identification and to exclude contaminants. Species identity of isolates was confirmed using an API 20C Aux yeast identification system (bioM rieux, Montalieu, France) . Additional tests to confirm the identity of species included morphological examinations such as the germ tube test, chlamydospore production test, and colony morphology observation.
All Candida isolates were tested for their in vitro susceptibility to voriconazole (Pfizer, Groton, CT, USA) and fluconazole (Pfizer) by BMD according to the approved standard reference method described in CLSI document M27-A3 .
Three laboratory technicians determined MICs independently at 24 h and 48 h after incubation at 35°C Because MIC values at 24 h and 48 h were found to be comparable (data not shown) we used only the 48 h readings for this analysis. The MIC values were based on consensus of at least 2 of the 3 technicians, otherwise the susceptibility test was repeated until a consensus was established. Interpretation of susceptibility was performed by applying the clinical breakpoints (CBPs) defined by CLSI document M27-A3 .
An inoculum of 1–5 × 106 cells/ml was used and applied to the agar plates (RPMI 1640 with 2% glucose) according to the manufacturer’s instructions. E-test strips (bioM rieux) were then applied. MICs were determined after 24 h and 48 h of incubation and interpreted according to CLSI document M27-A3 .
An inoculum of 1–5 × 106 cells/ml was used and disks applied to Mueller-Hinton agar with 2% glucose according to the manufacturer’s instructions (Bio-Rad Life Science, Marnes-la-Coquette, France). The plates were incubated at 35°C and read at 24 h and 48 h. Inhibitory zone diameters were interpreted according to CLSI document M44-A at the transitional point where growth abruptly decreased .
All methods were validated using quality control strains of C. albicans ATCC 90028, C. tropicalis ATCC750, C. parapsilosis ATCC 22019, and C. krusei ATCC 6258.
All statistical analyses were performed using IBM SPSS Statistics 21.0 for Mac (IBM Corp, Armonk, NY, USA). Descriptive statistics were used to present the data. The MIC values were reported as a range. P < 0.05 was considered significant. A Spearman correlation was used to determine agreement between BMD-MIC and ET-MIC values, and BMD-MIC values and DD assay diameters.
For the three-way comparison of BMD, ET, and DD, correlations were determined as recommended by the CLSI: (i) the definition of the occurrence of a very major error (VME) is when one strain is susceptible by the test methods (DD or ET), but is resistant by the BMD reference method; (ii) major errors (MEs) are defined as occurring when an isolate is found to be resistant by the test methods (ET or DD), but is susceptible by the BMD reference method; (iii) a minor error (mE) is indicated when an isolate is susceptible and dose dependent by one method, but susceptible or resistant by other methods. The total percentage agreement was calculated by the proportion of all test isolates that are in agreement, minus any errors, divided by the total number of test isolates.
Species distribution of isolates from candidemia
A total of 63 Candida isolates were tested including 32 (51%) C. albicans, 14 (22%) C. tropicalis, 12 (19%) C. parapsilosis, 4 (6%) C. glabrata, and 1 (2%) C. guilliermondii.
C. albicans isolates were highly susceptible to fluconazole (100%) and voriconazole (91%). No resistant strain of C. albicans was detected and only 3 strains were identified as susceptible-dose dependent (S-DD) against voriconazole.
C. tropicalis and C. parapsilosis were the majority (41%) of non-C. albicans Candida isolates. These species were also highly susceptible to fluconazole (93%–100%) and voriconazole (79%– 100%). Only one resistant strain (C. parapsilosis) was found against fluconazole and one resistant strain (C.tropicalis) was found against voriconazole. Antifungal susceptibilities of each Candida species are detailed in Table 1.
Antifungal susceptibilities of 63 Candida isolates.
|Antifungal agent||Candida||MIC range (μg/mL)||No. (%) of isolates indicated susceptibility category by CBPs|
|Fluconazole||C. albicans (32)||0.06–2||32(100)||0||0|
|C. tropic a H s (14)||0.06–64||13(93)||0||1(7)|
|C. parapsilosis (12)||0.125–4||12(100)||0||0|
|C. glabrata (4)||0.375–4||4(100)||0||0|
|C. guilliermondii (1)||1–1||1 (100)||0||0|
|Voriconazole||C. albicans (32)||0.06–2||29(91)||3(9)||0|
|C. tropic a I is (14)||0.03–1||11(79)||2(14)||1(7)|
|C. parapsilosis (12)||0.06–1||12(100)||0||0|
|C. glabrata (4)||1–4||2(50)||1(25)||1(25)|
|C. guilliermondii (1)||0.5–0.5||1 (100)||0||0|
Comparison of BMD, ET, and DD
The BMD-MIC values showed good correlation with ET-MIC values for fluconazole and voriconazole (r = 0.95; P < 0.001 and r = 0.94; P < 0.001 respectively). The BMD-MIC values were significantly correlated with inhibitory zone diameters observed for the DD assays for fluconazole and voriconazole (r = -0.47; P < 0.001 and r = -0.75; P < 0.001, respectively).
The total percentage agreement of the ET and DD determinations, compared with the standard reference BMD-MIC values for the two antifungal agents, exceeded 90% as summarized in Table 2.
Errors with the E-test and disk diffusion methods compared with broth microdilutions for fluconazole and voriconazole
|Method||Category||Fluconazole by BMD||No. of strains||Total agreement (%)||Voriconazole by BMD||No. of strains||Total agreement (%)|
The percentage agreement for the ET assays ranged between 92% to 98% and for DD between 92% to 98%. No VMEs were detected and only one ME was identified for a C. tropicalis isolate tested by DD with fluconazole. The mE detection rates ranged between 2% to 8% for ET determinations and 2% to 6% for DD determinations.
Determining the species distribution and susceptibility profiles of the yeast isolated from cases of invasive candidiasis is essential to guide physicians in the choice of antifungal agents in any empirical treatment, because these susceptibility profiles vary geographically and not all hospitals perform antifungal susceptibility tests routinely. Therefore, this information is crucial and currently lacking for Thailand and other places in South-east Asia. The last published data from Thailand were in 2002 . The present study provides an incidence and susceptibility profile for Candida isolated from blood in Thailand.
The predominant species was C. albicans (51%) and this was similar to that of previous studies in Southeast Asia and the ARTEMIS global surveillance study [6, 15-17]. The most common non-Candida albicans species in Thailand was C. tropicalis (22%) followed by C. parapsilosis (19%). These findings are consistent with data from other studies in Southeast Asia and China demonstrating that these two species are the predominant non-Candida albicans species. However in China and Malaysia, data have demonstrated a higher prevalence of C. parapsilosis than C. tropicalis [15-18]. Collectively, these results are in contrast with those from the USA and the ARTEMIS global surveillance study where C. glabrata was the most common non-Candida albicans species [6, 19]. These variations emphasize the importance of determining local epidemiologic patterns.
BMD as the standard antifungal testing method recommended by the CLSI can be difficult to perform in routine laboratory practice because of its complexity, the need of experienced laboratory technicians, and labor-intensive nature. By contrast, agar-based testing methods, (in particular DD, which has a ten-fold lower cost than ET) are commercially available, less expensive than BMD, easy to perform, and widely available, making them feasible as cost-effective screening methods. Our study demonstrated that BMD-MIC values showed good correlations with both ET-MIC and DD-MIC values. For the ET results, no VME was observed and the ME rates (92%–98%) were within the CLSI limits recommended in their document M-27A3 . The DD test results also demonstrated an excellent performance (92%–98%) and no VME was observed. Only 1 ME was found for fluconazole against C. tropicalis in DD. C. tropicalis MEs were also observed in a previous report ; therefore, laboratories should be aware of this possibility when performing DD with C. tropicalis species, and a standard BMD test may be warranted to confirm the antifungal susceptibility of C. tropicalis.
We found that the agar-based ET and DD assays are reliable alternatives to the standard reference BMD method for isolates that are susceptible to fluconazole and voriconazole, because they showed >90% agreement with the BMD method. The present study validates these agar-based methods for use as initial screening tools in Thailand [11, 12, 20]. A limitation of our study is that we only selected Candida isolates from blood; further study should include other sterile sites including cerebrospinal fluid, intravascular catheter samples or other sterile tissue samples for an improved validation of the agar-based methods.
In summary, DD is a valid alternative method to BMD for testing Candida susceptibility to fluconazole and voriconazole and can be performed as a cost-effective screening test in community settings in Thailand as a result of its economy and commercial availability.
We are very grateful to Prof. Arunaloke Chakrabarti, Postgraduate Institute of Medical Education and Research (PGIMER), Chandigarh, India for supplying the standard strains used in this study.
Our special thanks also go to all colleagues in the Mycology Unit, Department of Microbiology, King Chulalongkorn Memorial Hospital, and the Mycology Research Unit, Faculty of Medicine, Chulalongkorn University for providing the Candida isolates, and facilities and medical technologists from the Faculty of Allied Health Science, Chulalongkorn University, for their supportive team work.
Financial support: This work was supported by the 90th Anniversary of Chulalongkorn University Fund, Graduate School, Chulalongkorn University; the Ratchadaphisek-somphot Endowment Fund No. 168/54 and The Higher Education Research Promotion and National Research University Project of Thailand, Office of the Higher Education Commission (HR 1164A3). Antifungal agents were supplied by Pfizer (voriconazole and fluconazole). Mueller–Hinton agar was provided by Bio-Rad Laboratories.
Conflict of interest statement: Sansanee Lerdlitruangsin is an employee of MSD (Merck Sharp & Dohme) Thailand Ltd, a subsidiary of Merck & Co, Whitehouse Station, NJ, USA. No other authors have any conflict of interest to declare.
Authors’ contributions: AC, NW, DA, AT, and SL contributed to the acquisition, analysis, and interpretation of the data and drafted the article. VJ, WD, KK and SJ also drafted the article and critically revised it for important intellectual content. All authors read and approved the final manuscript.
Montagna MT, Caggiano G, Lovero G, De Giglio O, Coretti C, Cuna T, et al. Epidemiology of invasive fungal infections in the intensive care unit: results of a multicenter Italian survey (AURORA Project). Infection. 2013; 41:645-53.
Azie N, Neofytos D, Pfaller M, Meier-Kriesche HU, Quan SP, Horn D. The PATH (Prospective Antifungal Therapy) Alliance® registry and invasive fungal infections: update 2012. Diagn Microbiol Infect Dis. 2012;73:293-300.
Falagas ME, Apostolou KE, Pappas VD. Attributable mortality of candidemia: a systematic review of matched cohort and case-control studies. Clin Microbiol Infect. 2006;25:419-25.
Shorr AF, Gupta V, Sun X, Johannes RS, Spalding J, Tabak YP. Burden of early-onset candidemia: analysis of culture-positive bloodstream infections from a large U.S. database. Crit Care Med. 2009; 37:2519-26.
Pfaller MA, Diekema DJ. Epidemiology of invasive candidiasis: a persistent public health problem. Clin McrobiolRev. 2007; 20:133-63.
Pfaller MA, Diekema DJ, Gibbs DL, Newell VA, Ellis D, Tullio V, et al. Results from the ARTEMIS DISK Global Antifungal Surveillance Study, 1997 to 2007: a 10.5-year analysis of susceptibilities of Candida species to fluconazole and voriconazole as determined by CLSI standardized disk diffusion. J Clin Microbiol. 2010;48:1366-77.
Pappas PG, Kauffman CA, Andes D, Benjamin DK, Jr., Calandra TF, Edwards JE, Jr, et al. Clinical practice guidelines for the management of candidiasis: 2009 update by the Infectious Diseases Society of America. Clin Infect Dis. 2009; 48:503-35.
Pfaller MA, Diekema DJ. Progress in antifungal susceptibility testing of Candida spp. by use of Clinical and Laboratory Standards Institute broth microdilution methods, 2010 to 2012. J Clin Microbiol. 2012;50:2846-56.
Clinical and Laboratory Standards Institute. CLSI document M27-A3: Reference method for broth dilution antifungal susceptibility testing of yeasts; approved standard. 3rd ed. Wayne: Clinical and Laboratory Standards Institute; 2008.
Lee SC, Lo HJ, Fung CP, Lee N, See LC. Disk diffusion test and E-test with enriched Mueller-Hinton agar for determining susceptibility of Candida species to voriconazole and fluconazole. J Microbiol Immunol Infect. 2009; 42:148-53.
Sims CR, Paetznick VL, Rodriguez JR, Chen E, Ostrosky-Zeichner L. Correlation between microdilution, E-test, and disk diffusion methods for antifungal susceptibility testing of posaconazole against Candida spp. J Clin Microbiol. 2006; 44: 2105-8.
Matar MJ, Ostrosky-Zeichner L, Paetznick VL, Rodriguez JR, Chen E, Rex JH. Correlation between E-test, disk diffusion, and microdilution methods for antifungal susceptibility testing of fluconazole and voriconazole. Antimicrob Agents Chemother. 2003; 47: 1647-51.
Chen CY, Huang SY, Tsay W, Yao M, Tang JL, Ko BS, et al. Clinical characteristics of candidaemia in adults with haematological malignancy, and antimicrobial susceptibilities of the isolates at a medical centre in Taiwan, 2001–2010. Int J Antimicrob Agents. 2012; 40: 533-8.
Clinical and Laboratory Standards Institute. CLSI document M44-A. Method for antifungal disk diffusion susceptibility testing of yeasts; approved guideline. 2nd ed. Wayne: Clinical and Laboratory Standards Institute; 2004.
Foongladda S, Sakulmaiwatana P, Petlum P, Vanprapar N. Candida species, genotypes and antifungal susceptibility of Candida isolates from blood samples of patients at the largest tertiary care hospital in Thailand during 1999-2002. J Med Assoc Thai. 2004; 87:92-9.
Tan TY, Tan AL, Tee NW, Ng LS. A retrospective analysis of antifungal susceptibilities of Candida bloodstream isolates from Singapore hospitals. Ann Acad Med Singapore. 2008; 37:835-40.
Amran F, Aziz MN, Ibrahim HM, Atiqah NH, Parameswari S, Hafiza MR, et al. In vitro antifungal susceptibilities of Candida isolates from patients with invasive candidiasis in Kuala Lumpur Hospital, Malaysia. J Med Microbiol. 2011; 60:1312-6.
Xiao M, Fan X, Chen SC, Wang H, Sun ZY, Liao K, et al. Antifungal susceptibilities of Candida glabrata species complex, Candida krusei, Candida parapsilosis species complex and Candida tropicalis causing invasive candidiasis in China: 3 year national surveillance. J Antimicrob Chemother. 2015; 70:802-10.
Pfaller MA, Messer SA, Woosley LN, Jones RN, Castanheira M. Echinocandin and triazole antifungal susceptibility profiles for clinical opportunistic yeast and mold isolates collected from 2010 to 2011: application of new CLSI clinical breakpoints and epidemiological cutoff values for characterization of geographic and temporal trends of antifungal resistance. J Clin Microbiol. 2013; 51:2571-81.
Vandenbossche I, Vaneechoutte M, Vandevenne M, De Baere T, Verschraegen G. Susceptibility testing of fluconazole by the NCCLS broth macrodilution method, E-test, and disk diffusion for application in the routine laboratory. J Clin Microbiol. 2002; 40:918-21.