Fungal infections (mycoses) are classed into three groups: superficial fungal infections; subcutaneous fungal infections and invasive fungal infections. Invasive fungal infections can lead to fungaemia and these are a significant concern for healthcare professionals (Reimer, Wilson and Weinstein, 1997). These nosocomial infections are more frequently observed within intensive care units (ICU), than any other department. This is due to these departments caring for post-surgery patients who commonly have severe underlying illness or impaired immune systems (Alexander, Byrne and Smith, et al, 2007).
Long term antibiotic treatments, neutropenia caused by chemotherapy or underlying disease, immunosuppressant use, chronic illness and extended hospitalisation are all contributors to the increased incidence of fungal infections within the hospital environment (Toya, Schraunagel and Tzelepis, 2007). This is in addition to the use of invasive equipment such as intravascular catheters and ventilation equipment, as well as the use of antibacterial agents producing the ideal environment for widespread colonisation of these patients (Lass-Florl, Perkhofer and Mayr, 2010; Alexander, Byrne and Smith, et al, 2007).
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Mycoses usually develop slowly, taking months or years to become fully established infections that are capable of causing pathology. However, in immunocompromised patients fungal infections may be very aggressive, spreading rapidly to internal organs and causing mortality. The primary cause of systemic infections can be attributed to yeast isolates of the Candida species. Invasive Candida infections are the cause of up to 85% of mycoses, C. albicans being the documented cause of 60% of these Candida infections (Beers and Berkow, 1999).
Systemic infections of C. albicans have one of the highest mortality rates of all bloodstream infections (Flanagan and Barnes, 1998). Other species of Candida including C. glabrata, C. krusei, C. tropicalis and C. parapsilosis are isolated regularly from immunocompromised and surgical patients. This therefore highlights the importance of identifying Candida species causing infections and performing antifungal susceptibility testing (AST) on these. However, there is inconsistency in determining the susceptibility of clinically important Candida isolates to antifungal agents (Avolio, Grosso, Bruschetta, et al, 2009; Torok, Moran and Cooke, 2009).
Sensitivity and resistance to antifungal agents can be determined using the minimum inhibitory concentration (MIC). This is done by measuring the growth of an isolate in the presence of antifungal drug concentrations over a defined interval range, according to standard practice. The lowest drug concentration resulting in a considerable reduction or lack of growth of the isolate is the MIC for that agent (Lass-Florl, Perkhofer and Mayr, 2010).
A range of AST methods exist including macrodilution and microdilution, agar diffusion, disk diffusion and Etest. In addition, a number of antifungal testing systems are now commercially available, though these can produce variable results (Bertout, Dunyach and Drakulovski, et al, 2011 and Cuenca-Estrella, Gomez-Lopez and Alastruey-Izquierdo, et al, 2010).
The standardisation of AST was introduced by the Clinical and Laboratory Standards Institute (CLSI) and the European Committee on Antimicrobial Susceptibility Testing (EUCAST). This has changed the way AST is carried out and has enabled manual methods to be adapted to allow the development of both automated and semi- automated techniques in determining susceptibility to antifungals. The introduction of automated systems such as the Vitek2 has in turn allowed more reproducible results to be obtained (Cuenca-Estrella, Gomez-Lopez and Alastruey-Izquierdo, et al, 2010).
The primary problem faced by clinicians and microbiologists in the interpreting of in vitro AST is that MIC values do not directly correlate with response to antifungal therapy. This difference between in vitro and in vivo has resulted in the '90/60' ruling. This ruling suggests that susceptible pathogens will respond to treatment in 90% of instances, whereas resistant organisms respond 60% of the time (Cuesta, Bielza, Cuenca-Estrella, Larranaga, et al, 2010).
The aim of the learning contract is to compare 3 techniques of determining antifungal MIC of Candida species isolates. The 3 methods compared are the Etest, Vitek2 and Sensititre YeastOne YO8. The areas compared include ease of use in preparation and result interpretation together with the advantages and limitations of each method. This will be evaluated in order to determine the feasibility of implementing the Sensititre YeastOne YO8 system as a routine method of determining MIC within the Microbiology laboratory at Hull Royal Infirmary.
Clinical importance of Candida
Candida species are unicellular fungi characterised by round, oval or elongated cells (blastospores). Candida cell walls are made up of phosphorylated mannans, glucans and a small amount of chitin. The cells reproduce by budding which form daughter cells from the original, (mother), cell. The extension can be tubular and is the early stage of hyphae formation (germ tubes). This daughter cell may detach freely from the mother cell or remain attached, producing its own bud. During reproduction if the buds do not separate they form a chain of cells which have extended before budding (pseudohyphae). The morphology of yeasts can therefore either be budding and/or hyphal. Cells which form the pseudohyphae show a noticeable constriction between the cells (Gladwin and Trattler, 2008 and Campbell, Johnson, Philpot and Warnock, 1996).
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Figure1: Filamental hyphae and chlamydospores can be found in Candia albicans.Septum
In addition, some species of Candida, namely C. albicans produce true hyphae. Unlike pseudohyphae this is the development of branching hyphae composed of chitin that have been formed by one or more cells that have lengthened and divided by internal septum. C. albicans also produce chlamydospores (double walled cells), see figure 1 (Campbell, Johnson, Philpot and Warnock, 1996).candida
The presence of Candida species in the blood (Candidaemia) may represent disseminated candidiasis. However, this may also be due to colonisation of indwelling intravenous catheter devices (Kullberg and Filler, 2002). The number of patients within the hospital environment developing candidaemia is decreasing nationally with the Health Protection Agency (2009) reporting 1726 cases of isolation of Candida species from blood specimens, a 5% drop from 1814 cases in 2008. However, Edmond et al, (1999) report that Candida related nosocomial infections have the highest crude mortality rates at 40%. However, this rate may alter depending on the underlying complications or illness.
There are over 100 species of Candida with a number of species associated with candidaemia. It has been reported that, in Europe, although the most common cause of candidiasis is C. albicans, causing 49-53% of infection, there has been an increase in the occurrence of non-C. albicans related candidaemia. The most common being C. parapsilosis (11-21%), C. glabrata (10-12%), C. tropicalis (6-11%), C. krusei (1-9%) while 1-10% are other Candida species. Therefore rapid and accurate diagnosis of candidaemia is essential for clinicians to administer an appropriate therapy to their patients as quickly as possible (Kullberg and Filler, 2002 and Lundstrom and Sobel, 2001).
Isolation and Identification of Candida species
Figure 2: C. lusitaniae growth on Sabouraud's agar.A combination of morphological and biochemical techniques are required in order to investigate patient samples with suspected Candida infections. The clinical laboratory provides the growth requirements needed to ensure the successful isolation of the organism. The most commonly used medium for the inoculation of patient samples, for the isolation of Candida and other yeasts is Sabouraud's agar (SABS), figure 2. This media allows the growth of most pathogenic yeasts and are used for the isolation of both fungi and yeasts as the agar contains elements to aid their growth while also containing chloramphenicol which reduces the growth of bacteria and restrict growth on the media to fungi and yeasts (Health Protection Agency, 2005). Candida ID 2 (CAN 2) plates are a selective plate often used for the identification of Candida species. Enzymes present within C. albicans hydrolyse the hexosaminidase substrate within the agar causing the colonies of C. albicans to turn blue (Rousselle, Freydiere, Couillerot, et al, 1994).C:\Users\Leather\Pictures\Phone pics\IMG_0381.JPG
Other media used includes cornmeal agar for examination of morphological characteristics including the size and shape of the yeast cells. Cornmeal agar is used as it encourages the formation the morphological characteristics of hyphae, pseudohyphae and chlamydospores. These characteristics are useful in the identification of yeasts while the known ability to produce hyphae/pseudohyphae is also required when undertaking biochemical profile tests (Campbell, Johnson, Philpot and Warnock, 1996).
Biochemical tests available in identifying yeasts include understanding the metabolism and nutritional requirements during growth. The utilisation and fermentation of sugars by yeasts has allowed for the development of commercially available identification kits, such as the API 20C Aux (figures 3 and 4), which uses the yeasts ability to utilise the substrates available in the test strip as the sole carbon source (Campbell, Johnson, Philpot and Warnock, 1996).
Figure 3: Identification of C. albicans using the API 20C Aux method.
Figure 4: Identification of C. glabrata using API 20C Aux method.
The assimilation of the sugars allows growth to occur within specific cupules determining identification of the species by the specific profile obtained from this sugar metabolism. It is important that the morphology of the yeast, using cornmeal agar, is taken into account to complete this biochemical tests to avoid misidentification of organisms which have similar biochemical profiles. These morphological characteristics, metabolic processes and enzymologic characteristics are useful in the identification of the species causing infection (Campbell, Johnson, Philpot and Warnock, 1996).
Antifungal drugs: Mode of action and their use against Candida infections
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Due to its predominant medical importance, Candida has been investigated extensively by a number of studies in relation of the fungistatic and fungicidal properties of the limited number of antifungal agents available into determining the MIC of the various Candida species to these drugs. The determination of the MIC is vitally important for accurate and prompt clinical management and treatment of candidaemias (Avolio, Grosso and Bruschetta,et al, 2009). There are a limited number of antifungal drugs available for the treatment of fungal infections.
Antifungal drugs are designed to take advantage of the differences between human and fungal cell structure. This ensures that hazardous effects on host cells are limited (Madigan and Martinko, 2006). This is difficult as both host and fungal cells are similar at the molecular level, causing some drugs to producing side effects including life threatening conditions if incorrect dosages are used (Dismukes, 2000). Therefore determining the MIC of antifungal agents ensures that minimal damage will occur to the patient during treatment, while ensuring the correct treatment is being used for the infection.
A summary of the most common antifungals used and their mode of action are shown in table 1.
Table 1: Common antifungal agents used, the mode of action and their target areas.
Mode of action
Binds to ergosterol in the cell wall, disrupting membrane function increasing permeability causing cell death through ion leakage and loss of macromolecules.
Decreased ergosterol synthesis due to the inhibition of fungal cytochrome P450 enzymes.
Inhibits cytochrome P450 -mediated 14 alpha-lanosterol demethylation, preventing the conversion of lanosterol to ergosterol an essential constituent of the cytopasmic membrane of fungi.
Cell Wall Synthesis
Inhibits 1,3 β-D glucan synthase, an enzyme specific to fungi, which is used to form glucan polymers in fungal cell walls. Leading to rapid cell death.
Nucleic Acid Analog
DNA /RNA synthesis
Converted within fungal cells to 5-fluorouracil, 5-fluorodeoxyuridine monophosphate (DNA synthesis inhibitor) and fluorourdine triphosphate (RNA synthesis inhibitor. Inhibiting nucleic acid synthesis and further cell development.
(Adapted from Madigan and Martinko, 2006)
The limited number of antifungal drugs available for the treatment of Candida does not affect their action against the cells. However, treatment of Candida species may be problematic due to factors of stability absorption and drug stability. In addition, increases in the resistance of Candida species to these antifungals are also an increasing concern (Goering, Dockrell and Zuckerman, et al, 2008).
This increase in resistance to antifungal drugs is well documented with resistance to the azole agents including C. krusei. Due to an altered isoenzyme of the cytochrome P450, C. krusei is intrinsically resistant to fluconazole. While Pfaller, Diekema and Rinaldi, et al (2007) observed that a low number of C. albicans (1.5%) have developed a resistance to fluconazole due to prohylaxsis treatment of patients with fluconazole.
A number of studies have been conducted on the sensitivity patterns of Candidia species. These studies have concluded that there is an increase in the development of resistance to most of the common antifungals used for Candida species. This has included both C. lusitaniae and C. glabrata showing resistance and reduced susceptibility patterns in vitro from blood cultures of immunocompromised patients to amphotericin B (Moran, Sullivan and Coleman,2002).
Echinocandins including caspofungin have the highest success rate when dealing with Candida infections with resistance rare. However Hernandez, López-Ribot and Najvar, (2004)report of the development of caspofungin resistance in vitro, with analysis showing an increase in resistance found in isolates of C. albicans. However, the clinical relevance of this in vitro finding has yet to be determined as there is reduced correlation between in vitro MIC and clinical implications..
The prophylaxis use of antifungal drugs has resulted in a number of Candida species developing some resistance, to most of the commonly used antifungal agents. Consequently this has allowed the development of fungal pathogens which were previously non pathogenic causing diseases in immunocompromised patients (Madigan and Martinko, 2006). Therefore, determining minimum inhibitory concentration of these antifungal agents may aid laboratories and clinicians observe these differences in antifungal patterns signalling a development of antifungal resistance for that organism.
Methods of detecting Minimum Inhibitory Concentration (MIC)
The importance of a clinical laboratory in testing for susceptibility is the ability to produce accurate results within a reasonable turnaround tine in order to aid clinical treatment. There are a number of methods available including the fully automated Vitek 2 system, Etest strip diffusion and Sensititre YeastOne microdilution YO8 systems.
Figure 5: The Vitek 2 (Biomerieux) is a fully automated identification and susceptibility testing system.C:\Users\Leather\Pictures\Phone pics\IMG_0385.JPG
The Vitek 2, Biomerieux, (figure 5) is a fully automated method which spectrophotometrically determines yeast growth and is capable of both fungal identification and determination of MIC using the YST identification card which compares the biochemical profile of the test isolate with a large database of known species (Pfaller, et al, 2007). The system also features the AST-YS01 susceptibility testing card which includes amphotericin B, fluconazole, flucytosine and voriconazole. This card is a mini version of the microdilution method utilising 64 wells containing antifungal agents at varying dilutions. The MIC is determined through the use of the systems advanced expert system software which interprets susceptibility results established by a decrease in light measured by an optical scanner producing the MIC values for each antifungal agent (Cuenca-Estrella, et al, 2010)
The Etest manufactured by AB Biodisk is an agar based quantitative diffusion method using pre-coated strips with predefined concentration gradients of antifungal agents. These non-porous strips are based on a 15 two-fold dilution found in traditional macrodilution MIC testing methods. A number of antifungal agents are available in this format including Amphotericin B (range 0.002-32 µg/ml); Fluconazole (range 0.016-256µg/ml); Voriconazole (range 0.002-32ug/ml) and Caspofungin (0.002-32µg/ml). The determination of MIC is made after 24-48 hours incubation when visible growth showing a symmetrical inhibition ellipse along the strip is exposed (figure 6), this ellipse intersects the strip producing the MIC value (Kahlmeter, 2003).
Figure 6: Amphotericin B Etest showing the noticeable zone of inhibitionInterpretation of the MIC is dependent on the antifungal agent being used. MIC should be interpreted for Amphotericin B at complete or 100% inhibition of all growth. 80% inhibition is recorded for agents such as the echinocandin (Caspofungin) and the azoles (Fluconazole and Voriconazole) this is due to the trailing effect which shows a marked decrease in growth density (Kahlmeter, 2003).
This technique has enabled clinical laboratories the ability to produce the MIC in-house susceptibility testing with reproducibility to ± 1 dilution step. However, this method requires a suitable range of experience, due to the presence of microcolonies within the inhibition ellipse over several dilutions, method order to ensure confident results (Kahlmeter, 2003).
The Sensititre YeastOne YO8, is a microdilution method based on the CLSI standards. Each test consists of a disposable 96 well microtitre tray containing dried serial dilutions of eight antifungal agents. These include Posaconazole (range 0.008-8µg/ml), Amphotericin B (range 0.008-16µg/µml), Fluconazole (range 0.125-256µg/ml), Itraconazole (range 0.008-16µg/ml), Ketoconazole (range 0.008-16µg/ml) Flucytosine (range 0.03-64µg/ml), Voriconazole (range 0.008-16µg/ml) and Caspofungin (range 0.008-16µg/ml) in individual wells (figure 7).
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Figure 7: The wells of the YeastOne YO8 produce a colour change (from blue to pink) with the presence of yeast. The MIC results are indicated by a colour change from blue to pink with the growth of yeasts via the inclusion of Alamar Blue, a colorimetric indicator. The MIC is the lowest concentration of antifungal agent that substantially inhibits the growth of the organism, which results in the lack of colour change from blue to pink.
This colour change improves ease of end point readability and reproducibility with limited training. Each test plate also includes a positive control well in well A1 this ensures quality control measures are maintained. Avolio, et al, (2009) suggested that the use of Sensititre YeastOne direct for a Candida positive blood culture could reduce the turnaround time and provide rapid antifungal MIC determination which may ensure prompt clinical treatment for patients with candidaemia.
[Quality assurance is an important aspect of laboratory practice and processing patient samples to ensure confidence in the results produced. This includes manufacturer quality control, internal quality assurance (IQA) and external quality assurance (EQA). EQA is the comparison of a laboratories result with the predetermined standards, which are maintained by the National External Quality Assessment Service (NEQAS). Microbiology Laboratory at Hull Royal Infirmary partakes in this quality assurance scheme, with samples received treated in the same way as patient samples. This service has a number of benefits of participation including providing laboratory managers an insight into performance, ensures confidence on all results and aids with improvement of laboratory service for the user. The method of internal quality control was implemented with a patient isolate being investigated twice to determine the reproducibility of the results. Furthermore, the inclusion of two NEQAS samples provided samples with known sensitivity patterns to further ensure quality control (See Appendix 1).
The study had a number of quality control measures attached to them at different points of the process to ensure confidence in the results. The API 20C Aux has the provision of a negative control cupule for comparison of positive and negative growth during the identification process. The National Collection of Pathologic Fungi, (NCPF) quality control strain C. albicans was used in the investigation of hyphal growth on cornmeal agar and used as a basis for comparison of the test isolates. Both the Vitek 2 and Sensititre YeastOne YO8 employ the use of positive growth well to ensure confidence in the MIC results, with the failure to produce positive growth within the well resulting in a terminated test in the case of Vitek 2 or unattainable MIC levels within the YeastOne system. The inclusion of duplicate testing of one specimen allowed for the provision of an IQA, to further ensure confidence in the systems and to reflect reproducibility of results.
Specimens and Identification
Seventeen Candida positive blood culture samples (with one sample acting as an internal quality control) obtained from the BacT/ALERT automated detection system, plus 2 NEQAS freeze dried samples were inoculated onto Sabouraud's agar plates then incubated aerobically for 24 hours at 37°C to produce single colonies. Identification of yeast presence was determined via the use of the Grams staining method, for the presence of budding yeast.
Species identification was obtained through the use of the API 20C Aux identification strip, following manufacturer protocol and incubated at 30°C for 72 hours, with occurring at 48 and 72 hours. The inoculation of cornmeal agar plates was performed to complete the final test of the API 20C Aux and aid identification by determining the presence of hyphae/pseudohyphae and chlamydospores.
Minimum Inhibitory Concentration (MIC) Methods
The Etest method was performed using one RPMI agar with 2% glucose buffered with morpholinepropanesulfonic acid (MOPS) plate per antifungal agent used. Inoculum isolates were homogenised in 0.9% sterile saline, achieving a 0.5 McFarland turbidity which was adjusted using a spectrophotometer. The suspension was inoculated covering each plate in three directions using a sterile swab, any excess moisture was allowed to absorb, for 15 minutes, before addition of the Etest strips. MIC readings were obtained visually after 24-48 hours incubation at 37°C. MIC was determined as the value where endpoints for caspofungin, voriconazole and fluconazole were at 80% inhibition with MIC for Amphotericin B measured at 100% inhibition.
Vitek 2 investigation isolates were homogenised in a sterile polystyrene test tube with saline to a 1.8-2.2 McFarland standard adjusted using the Vitek 2 DensiCHECK, then added to the smart cassette carrier, with the addition of a yeast identification card and a second empty sterile polystyrene test tube and antifungal susceptibility test card for each organism. These were then added to the VITEK 2 instrument where suspensions were diluted, cards filled and incubated by the analyser then read automatically by the system software.
The YeastOne YO8 method was performed after homogenising each test isolate in sterile water achieving 0.5 McFarland turbidity adjusted using a spectrophotometer. 20µl of the suspension was then transferred to an 11ml YeastOne inoculation broth and mixed well. 100µl was then transferred to each well of the antifungal agent tested. Colony growth control plates were included by removing 10µl from the positive control well and inoculate onto SABS agar which when diluted correctly should produce 15-80 colonies. All wells were covered with the adhesive seal provided avoiding creases and incubating at 37°C for 24 hours.
All MIC readings was determined for each of the methods using values set by CLSI as seen in table 2, below.
Table 2: CLSI standard values for MIC of Candida spp. to each antifungal agent tested
Susceptible Dose Dependent (SSD)
0.03 - 2
* Isolates of C. krusei assumed to be intrinsically resistant so not interpreted using this scale.
The following results were observed from the 20 Candida species isolated in order to compare the methods used of determining antifungal MIC (The full list of results can be found in appendix 2).
The identification results, table 3, show the number of each species isolated from the total number specimens. These identifications are needed to determine the antifungal MIC levels for each isolate.
Table 3: Species isolated from 20 blood culture specimens identified using API 20C Aux.
Candida species isolated
Number of total isolates (%)
Figure 8 (below), shows an example of the zone of inhibition used to determine the MIC when using the Etest. The MIC for the specimen MM425038 was 0.125µg/ml. These zones of inhibition were determined with a 100% inhibition for Amphotericin B and 80% inhibition for Caspofungin, Fluconazole and Voriconazole.C:\Users\Leather\Pictures\Phone pics\IMG_0384.JPG
MIC = 0.125µg/ml
Figure 8: The MIC for specimen MM425038 was recorded at 0.125µg/ml for Amphotericin B at 100% inhibition.
Results for the YeastOne YO8 method were observed due to a colour change in the wells as shown below. MIC levels were read as the first blue or well with no positive growth. The results also show the trailing effect detailed for fluconazole (figures 9 and 10, Row C) as reported in a number of MIC studies.
Figure 9: Sample MM425038, C. lusitaniae showing a trailing effect of Fluconazole (arrowed) and also MIC of <0.008µg/ml for Voriconazole (Row G).
Trailing effectC:\Users\Leather\Pictures\Phone pics\IMG_0351.JPG
Figure 10: Specimen MM250815 II, C. albicans showing skips in wells on rows G and H (arrowed).
Specimen MN38703 (figure 11) was found to be contaminated with Gram negative bacilli with the wells, producing clear to light pink wells as opposed to the unmistakable blue or red/pink wells and was deemed unviable in determining MIC.
Figure 11: Specimen MN38703 resulted in a plate which was unable to read due to contamination with bacteria.C:\Users\Leather\Pictures\Phone pics\IMG_0356.JPG
A selection of Vitek 2 results can be found in appendix 3. These results were obtained using the Vitek 2 Advanced Expert System, which identifies the species of Candida and determines the MIC based on this phenotypic identification. Relevant areas to notice were the modification of the fluconazole sensitivity reported as resistant agreeing with
As previously reported Kullberg and Fuller (2002) the most common species isolated was identified as being C. albicans as well as a high number of non C. albicans species isolated including C. glabrata. During this study all identification methods produced high confidence in results with levels >95%, while the identification of hyphae and pseudohyphae also correlate with each isolate identity. However, specimen MN38278, was a C. albicans isolate which produced true hyphae without the production of chlamydospores during cornmeal agar examination. However, manufacturer guidelines state that isolates of C. albicans which have undergone a number of subcultures may produce this anomalous result on cornmeal agar.
As with all scientific techniques there were a number of advantages and limitations observed with each method. The ease of preparation for all three methods was compared with all techniques being relatively simple to prepare and use. However, Vitek 2 was considered to have the advantage as it is less labour intensive with dilution and inoculation performed by the analyser. YeastOne YO8 was found to be more labour intensive in inoculation of the Sensititre trays due to the number of wells. Nevertheless, this issue was raised during the study and a possible solution discussed. Consequently, if this system was to be routinely used, a multi-head pipette system could be obtained to resolve this issue.
Ease of interpretation of MIC for each method was very different, with the most simple being the Vitek 2 system as all interpretation was carried out by the system software. The YeastOne YO8 system was found to be a quick and easy method of interpreting MIC as it produced an obvious colour change for the different levels of MIC, with the trailing effect of fluconazole producing a purple coloration of the wells. The most difficult method to interpret was the Etest this was due to individual species growth levels and the need to determine MIC depending on the 100% or 80% inhibition levels which were subjective readings as discussed by Araj et al, (1998). This is further supported as the fluconazole Etest NEQAS result reported by the laboratory for specimen MM251403 was determined as intermediate, whereas this study found it to be resistant using the same Etest method. This inconsistency is a result of the subjective interpretation by different users.
The cost per isolate for each test is also a factor when implementing a system for routine use. The price per method were discussed with the laboratory manager and it was noted that to test four antifungals against an isolate the Etest was the most expensive at approximately £20, while the Sensititre YeastOne YO8 system was the next expensive at approximately £12 per isolate. However, this method allowed the testing of eight antifungals commonly used clinically, the most inexpensive test per isolate was the Vitek 2 at approximately £7 per isolate, for 4 antifungals, not including caspofungin. The major disadvantage of this system is the initial cost of the analyser which is required. Therefore, the use of the YeastOne YO8 system may be better value for money and allow alternative antifungal treatment for patients.
The YeastOne YO8 control growth plate for MM250582 produced 97 colonies, a larger number than the 15-80 optimal range of colonies, as recommended by the manufacturer. However the MIC levels found for the isolate was still comparable with the other two methods producing results that were still within the susceptibility ranges provided by CLSI.
The Vitek 2 AST-YS01 card reading for specimen MM251402, a NEQAS isolate of C. kefyr, failed to grow within the positive control well for MIC interpretation and was therefore terminated. This made it impossible to compare all three methods. As this was a NEQAS sample with a known susceptibility pattern the report shows that both the YeastOne YO8 and Etest sensitivity results for all five antifungal agents obtained from this study correlated with the external examining body results. This includes the intermediate result obtained for Flucytosine using the YeastOne YO8 system, an antifungal agent that is not routinely investigated by Hull Royal Infirmary.
The second NEQAS specimen MM251403, was an isolate of C. krusei which is intrinsically resistant to fluconazole (Kullberg and Fuller, 2002). This suggested that all MIC readings falling within the sensitive and intermediate levels are disregarded and reported as resistant. During this study all three systems correctly identified this fluconazole resistance pattern with the Vitek 2 Advanced Expert System modifying the result printed as it automatically identifies detects resistant phenotypes and corrects the results accordingly. Both the Vitek 2 and YeastOne YO8 systems also correctly identified an intermediate result for Flucytosine, the expected result for this sample.
MIC results for specimen number MN38703 were disregarded as this specimen was a mixed culture of both C. parapsilosis and Gram negative bacilli. This was identified via the YeastOne YO8 system as all wells produced a pale pink colour as opposed to positive control wells of all other isolates. As recommended a selection of wells were subcultured onto Columbia blood agar plates and incubated for 24 hour and subsequent growth showing a mix of both yeast cells and Gram negative bacilli using the Grams staining method.
Specimen number MN38488, an isolate of C. glabrata has shown a difference in susceptibility results against fluconazole with the Etest producing a sensitive MIC result, the Vitek 2 system produced an intermediate result while the YeastOne YO8 system produced a resistant reading. Consequently, there would be the need to repeat the tests for this isolate. However the YeastOne YO8 result agrees with reports that C. glabrata is intrinsically resistant or can rapidly develop resistance to Fluconazole (Heitman, 2006) and Pappas, Rex and Sobel, et al, 2004).
Specimen numbers MM425152 I and MM425152 II were used as an IQA specimen and therefore should have produced comparable results. This is true for both the Vitek 2 and Etest methods with both sets of results having exactly the same MIC readings. However, the YeastOne YO8 method had higher MIC readings for specimen number MM425152 II, this may have been caused by the need for this Sensititre tray requiring a further 24 hour incubation due to the positive control well failing to turn positive within the 24 hour period required for specimen MM425152 I. Also MIC results for both voriconazole and caspofungin were undetermined using the YeastOne YO8 as there were skips in the wells, caused by creasing of the protective film covering, which deemed theses MIC as failures requiring them to be repeated.
This study has highlighted the importance of sensitivity testing of Candida infections and the difficulties faced during processing and interpretation of the results for the successful treatment of patients with candidaemia. It has also shown the need for reliable identification, through a number of morphological and biochemical techniques.
In conclusion, comparison of the Vitek 2 , Etest and Sensititre YeastOne YO8 methods of determining antifungal MIC, has shown that each of the techniques have advantages and limitations associated with them. However, the Vitek 2 YST and AST-YS01 card systems are considered to be superior to the Etest. This is due to them offering a quick and easy to use testing method that provides antifungal sensitivity results for the treatment of Candida species, which are easy to interpret and are reproducible. In addition, they offer a reduced cost per isolate for routine testing of clinically important Candida compared to the Etest.
However, the initial outlay in cost of purchasing the Vitek 2 analyser is a major disadvantage as many laboratories are unable to fund these large items. Therefore, the use of the Sensititre YeastOne YO8 system is a cheaper alternative to both the Vitek 2 and the Etest in the determining of antifungal MIC. This is due to the ease to use and cost per isolate being substantially cheaper than the Etest. Furthermore, this system contains a higher number of antifungal agents providing alternative treatments to those that are routinely tested within the laboratory at present.
The inclusion of the Sensititre YeastOne YO8 system in this learning contract has prompted the Microbiology laboratory at Hull Royal Infirmary to purchase a number of YeastOne YO8 kits to run alongside the traditional Etest and investigate further the feasibility of implementing this system as a routine method of determining the antifungal MIC of clinically important Candida isolates.