ul. Debinki 7, Gdansk, Poland 3 Department of Clinical Microbiology and Immunology, The Children s Memorial

Transcription

1 Acta Microbiologica et Immunologica Hungarica, 61 (2), pp (2014) DOI: /AMicr EVALUATION OF POSSIBILITIES IN IDENTIFICATION AND SUSCEPTIBILITY TESTING FOR CANDIDA GLABRATA CLINICAL ISOLATES WITH THE INTEGRAL SYSTEM YEAST PLUS (ISYP) PIOTR SZWEDA 1*,KATARZYNA GUCWA 1,LUKASZ NAUMIUK 2, EWA ROMANOWSKA 3,KATARZYNA DZIERZANOWSKA-FANGRAT 3, ANNA BRILLOWSKA-DABROWSKA 4,IWONA WOJCIECHOWSKA-KOSZKO 5 and SLAWOMIR MILEWSKI 1 1 Department of Pharmaceutical Technology and Biochemistry, Faculty of Chemistry, Gdansk University of Technology, ul. G. Narutowicza 11/12, Gdansk, Poland 2 Department of Clinical Microbiology, Hospital of the Medical University of Gdansk, ul. Debinki 7, Gdansk, Poland 3 Department of Clinical Microbiology and Immunology, The Children s Memorial Health Institute, Aleja Dzieci Polskich 20, Warsaw, Poland 4 Department of Microbiology, Faculty of Chemistry, Gdansk University of Technology, ul. Debinki 7, Gdansk, Poland 5 Department of Microbiology and Immunology, Pomeranian Medical University, Powstancow Wielkopolskich 72, Szczecin, Poland (Received: 16 December 2013; accepted: 26 April 2014) The aim of this study was to evaluate possibilities of correct identification and susceptibility testing of C. glabrata clinical isolates with Integral System Yeast Plus (ISYP). For species identification, as the reference method, API Candida test and species-specific PCR reactions were used. The potential of antifungal susceptibility testing by the ISYP test was compared with the Sensititre Yeast One. Whilst the reference methods confirmed that the received population (n = 65 isolates) represented only C. glabrata, identification with the ISYP system showed correct data only in the case of 18 strains tested (27.7%). Species identification of the other 47 strains with the ISYP test was not possible at all. Significant differences were also observed for drug susceptibility testing carried out by the ISYP and the Sensititre Yeast One. The highest level of disagreement in classifying strains as resistant or susceptible estimated, as 73.9% and 40.0%, was observed for itraconazole and amphotericin B, respectively. Satisfactory results were only obtained for 5-fluorocytosine with 93.8% agreement between both methods. In our opinion the idea of the ISYP system is certainly good. The combination of identification ability and drug susceptibility testing in one test is very im- * Corresponding author; piotr.szweda@wp.pl /$ Akadémiai Kiadó, Budapest

2 162 SZWEDA et al. portant, especially from a clinical point of view. However, the current version of the ISYP has many disadvantages. We would like to encourage the manufacturer to make an effort and develop a new, more accurate version of the test. Keywords: C. glabrata, species identification tests, susceptibility testing Introduction During the last two decades, the occurrence of fungal infections caused by Candida spp., has increased dramatically, mainly due to a rise in the number of immunocompromised patients [1, 2]. Currently, Candida spp. are ranked as the fourth most common cause of nosocomial bloodstream infections in the US [3, 4]. The mortality rate associated with systemic Candida infections reaches approximately 35 50%, and annual treatment costs exceed US$10 billion in the US alone [5]. In most cases, Candida infections are derived from the individuals own endogenous reservoir when the host represents certain risk factors, such as immunosuppressive and cytotoxic therapies, treatment with broad spectrum antibiotics, AIDS, diabetes and drug abuse. C. albicans is the most common fungal pathogen in humans, although recently a shift towards systemic infections by non-albicans species has been reported. In a tertiary paediatric hospital in Poland the prevalence of non-albicans species increased from 12.5% in 2000 to 70% in 2010 [6]. In the case of some non-albicans species, e.g. C. glabrata and C. krusei, resistance to available antifungal agents used as a first-line antifungal therapy, especially fluconazole, is very common [7]. Therefore, correct identification and susceptibility testing of the infecting organism has become essential to guide antifungal therapy. There are no doubts that in recent years there has been significant progress in the diagnosis of fungal infection. However, most of the research in this area has focused on using the newest methods including PCR [8 10] and MALDI-TOF MS [11, 12]. Due to the high cost of equipment required for these techniques, they are not accessible for most clinical laboratories. Therefore, it is still necessary to improve the conventional diagnostic techniques which may also be used in less affluent hospitals. There are many problems in determination of drug susceptibility in Candida spp. The reference microdilution method for determination of MIC of antifungal agents developed by The Clinical Laboratory Standards Institute (CLSI) [13 15] is time-consuming and laborious. An interesting solution for both problems (identification and susceptibility testing of yeast pathogens) has recently been proposed by Liofilchem S.r.l. Roseto D.A. Teramo (Italy), who developed the Integral System Yeast Plus (ISYP) sys-

3 IDENTIFICATION AND SUSCEPTIBILITY TESTING FOR CANDIDA GLABRATA WITH ISYP 163 A B Figure 1. Comparison of susceptibility testing with Sensititre and Integral System Yeast Plus tests for fluconazole (Panel A) and flucytosine (Panel B). According to the reference microdilution method for susceptibility testing (CLSI) breakpoint, the MIC for fluconazole is 64 µg/ml and 32 µg/ml for flucytosine. As shown in Panel A, strains with fluconazole MIC of 64 µg/ml are misclassified as sensitive in ISYP. In case of flucytosine, classification in both systems is in concordance. A half concentration of the breakpoint MIC of tested agent is necessary for correct classification strains as resistant in ISYP tem. The aim of our study was to evaluate the quality of this system by comparison with other methods currently applied for yeast identification and drug susceptibility testing. Materials and Methods As a model for our research, a collection of 65 clinical isolates, initially classified as C. glabrata was used. The isolates were obtained from various physiological sites from patients from three Polish hospitals; the Children s Memorial

4 164 SZWEDA et al. Health Institute in Warsaw (17 isolates), the Medical University of Gdansk (38 isolates) and the Pomeranian Medical University of Szczecin (10 isolates). Species identification was confirmed by two phenotypic methods: growth on Chromagar Candida Lab-agar plates (Biocorp), API Candida test (biomérieux), as well as by PCR reaction. On Chromagar Candida Lab-agar plates C. glabrata isolates revealed a violet colour. Using the API Candida test a characteristic pattern of sugar assimilation was obtained, with only two positive fermentation results for glucose and trehalose (which is specific for this species). The molecular identification was carried out according to the method developed by Brillowska and co-workers [16], with slight modifications. DNA was extracted with a Genomic Mini AX Yeast Kit (A&A Biotechnology, Poland) according to the enclosed protocol. Individual PCR amplifications contained 1 µl of prepared DNA template and 19 µl of the following PCR master mixture: 10 µl of 2 PCR Mix Plus High GC (A&A Biotechnology, Poland), (Taq DNA polymerase 0.1 U/µL, MgCl 2 4 mm, dntps [datp, dgtp, dctp, dttp], 0.5 mm of each, plus factors increasing the specificity of PCR reaction, red dye, loading buffer), 1 µl of each of primers solutions (10 µm) and 7 µl of distilled, nuclease-free water. The DNA sequences of the primers used were as follows: forward 5 -GAGTGGTATGACG AGCAATGGT-3 and reverse 5 -TGTATTGAAGATTCCCTCTCATATATC-3. Amplification conditions were as follows: 4 min of initial denaturation at 94 C, 60 s of denaturation at 94 C, 30 s of annealing at 57 C, and 60 s of primer extension at 72 C repeated for 32 cycles and 10 min of final extension at 72 C. The PCR products (272 bp) were detected on 1.5% agarose gel with addition of ethidium bromide. Susceptibility testing of the isolates was performed with a customized test Sensititre Yeast One (TREK Diagnostic Systems, Cleveland, OH, USA). Pure cultures of tested yeast strains were cultured on Sabouraud dextrose agar at 37 C for 24 h. Separate colonies were suspended in sterile water to obtain an optical density of OD 660 = 0.1. In the next step 20 µl of the suspension was diluted in 11 ml of Yeast One Broth and 100 µl of the inoculum was transferred to the wells of microtiter plates containing dried serial dilutions of nine antifungal agents: amphotericin B (range µg/ml), fluconazole (range µg/ml), posaconazole (range µg/ml), voriconazole (range µg/ml), itraconazole (range µg/ml), 5-fluorocytosine (range µg/ml), anidulafungin (range µg/ml), micafungin (range µg/ml), caspofungin (range µg/ml). The wells contained Alamar Blue as a colorimetric indicator, which greatly improves the end-point readability by a col-

5 IDENTIFICATION AND SUSCEPTIBILITY TESTING FOR CANDIDA GLABRATA WITH ISYP 165 our change from blue to pink. Plates were covered with sealing foil and incubated at 37 C for 24 h. MIC values were established as the first well where no colour change had been observed. Susceptibility to fluconazole was also confirmed by reference microdilution method [14, 15]. Isolate suspension at OD 660 = 0.1 was established in sterile water and 50-fold diluted in RPMI-1640 medium (Sigma Aldrich, USA). A volume of 100 µl of prepared inoculum was transferred to the wells of a vertically positioned microtiter plate containing double dilutions of fluconazole (in the range of concentration of µg/ml) in RPMI-1640 medium. Following incubation at 37ºC for 24 h the determination of the MIC values of the fluconazole was performed by measuring the absorbance at 531 nm using a Victor 3 microplate reader (Perkin Elmer, Waltham, USA). The MIC value of fluconazole was defined as the lowest concentration yielding inhibition of growth equal to or higher than 90% of growth control. Identification and antifungal susceptibility testing with the ISYP was carried out according to the manufacturer s protocol. Briefly, the yeasts were grown at 37 C for 24 h on non-selective Sabouraud dextrose agar. Two or three separated colonies were selected and suspension A was prepared in sterile water (OD 660 = 0.1). Then a xylose disc was added to the ninth well of the ISYP plate and 200 µl of suspension A was transferred to twelve wells containing different sugars, and to the thirteenth with a chromogenic substrate. In order to test drug susceptibility, 20 µl of suspension A was added to 4.5 ml of sterile saline solution and suspension B was obtained. Two hundred microliters of suspension B were transferred to each of the remaining wells containing dried antimycotics. The final concentrations of tested antimycotics in the wells of the plates were as follows: nystatin (1.25 µg/ml), amphotericin B (2.0 µg/ml), 5-fluorocytosine (16.0 µg/ml), econazole (2.0 µg/ml), ketoconazole (0.5 µg/ml), clotrimazole (1.0 µg/ml), miconazole (2.0 µg/ml), itraconazole (1.0 µg/ml), voriconazole (2.0 µg/ml), fluconazole (64.0 µg/ml). The wells, except the one with the chromogenic substrate, were covered with two drops of vaseline oil. Plates were incubated at 37 C for 48 h. A colour change from red to yellow indicated resistance to the antimycotic in the applied concentration. A red to orange colour change indicated an intermediate sensitivity, whereas no colour change showed that the examined isolate was sensitive to the concentration of the antimycotic in the well. Both tests, the ISYP and the Sensititre Yeast One, were performed using the same suspension of isolates at the same time to be sure that results are not affected by random variables. Identification of the yeast was based on assimilation of various sugars and the consequent ph change in the well, which led to a colour change.

6 166 SZWEDA et al. Results and Discussion The results of the three mentioned methods: yeast growth on Chromagar Candida Lab-agar plate, API Candida test and PCR, confirmed that the received population represented only C. glabrata species. Unfortunately, in most cases an attempt to determine the species of the strains tested with the ISYP system was not successful (Table I). It is characteristic of C. glabrata that it is able to ferment only Table I Results of species identification test Assimilated sugars Tre + Glc Tre + Glc + Xyl Tre + Glc + Dul Tre + Glc + Xyl + Dul Number of strains Percentage Tre trehalose; Glc glucose; Xyl xylitol; Dul dulcitol two sugars, glucose and trehalose, and this feature is the basis of identification. The ability of sugar assimilation is observed as a colour change in a well from brown to yellow. Correct results were received only in 18 cases (27.7%) out of 65 examined isolates. In the case of other tested strains positive results for sugar assimilation were also observed for xylose and/or dulcitol (Table I). From the combination of positive and negative reactions, a numerical code was calculated and the identification of the yeast under examination was made by consulting the codebook. For isolates with positive results for xylose and/or dulcitol assimilation, the obtained numerical code did not fit any species from the codebook, which is why species identification was not possible. Significant differences were also observed for drug susceptibility testing performed by the ISYP and the Sensititre Yeast One (Table II). Because several comparative studies of the Sensititre Yeast One test with the CLSI method [17] and E-tests [18] have shown favourable results, we decided to use this product for evaluation of possibilities of analysis of drug susceptibility of Candida spp. with the ISYP test. Additionally, the investigated population of C. glabrata isolates was tested for fluconazole susceptibility with the reference method recommended by CLSI, and the obtained results were in agreement with the results achieved by the Sensititre Yeast One. In both tests, the Sensititre Yeast One and the ISYP, there is a group of five repeated antimycotics: amphotericin B, 5-fluorocytosine, itraconazole, voriconazole and fluconazole. The Sensititre Yeast One test allows determination of the

7 IDENTIFICATION AND SUSCEPTIBILITY TESTING FOR CANDIDA GLABRATA WITH ISYP 167 Table II The results of susceptibility testing Antimycotic AB FC IZ FZ VOR A number of resistant strains according to Sensititre (17)* B number of resistant strains according to Integral C number of resistant strains according to both tests * 1 10 D number of sensitive strains according to both tests E Percent of agreement = ((C + D)/65) 100% F Percent of disagreement = 100% E AB amphotericin B; FC flucytosine; IZ itraconazole; FZ fluconazole; VOR voriconazole * In parentheses are presented the number of strains growing in media containing itraconazole in the concentration of ³1 µg/ml, as it was analysed in ISYP. In fact recommended by CLSI susceptibility breakpoint for itraconazole (1 µg/ml) should be taken into account (the number of resistant strains 47). In this case resistant strains with MIC =1 µg/ml grew only in the medium containing lower concentration of this agent in Sensititre test in the wells containing itraconazole in concentration of 0.5 µg/ml. These strains (n = 30) in ISYP test are misclassified as sensitive. exact value of MIC (Minimal Inhibitory Concentration), while in the ISYP test it is only possible to check if the strain is resistant or sensitive to the drug. However, this is not completely correct. The manufacturer suggests that the wells contain such an amount of agents, which after adding the inoculum creates the concentration at which only a resistant strain (according to CLSI guidelines M27-A3) would be able to grow. This rule would be true if the final concentration of the agents would be equal to half of that recommended by CLSI breakpoint MIC values. For example, in the case of fluconazole and itraconazole, the final concentration in wells is the same as the breakpoint for these agents. As a consequence, some strains recognized as susceptible in the ISYP test in fact can be resistant, with the MIC value close to the breakpoint (Fig. 1). Moreover, it should be noticed that on December 2012, the M27-S4 [19] supplement was communicated which includes the new breakpoints for fluconazole and other antifungal agents. According to the new document C. albicans, C. tropicalis and C. parapsilosis are classified as susceptible when their MIC value for fluconazole is 2 µg/ml. The fluconazole resistance breakpoint for mentioned Candida species is 8 µg/ml. Interestingly the fluconazole resistance MIC breakpoint for C. glabrata has not been changed (64 µg/ml) in the new CLSI document (M27-S4). Despite this, the current version of ISYP is not a useful tool for analysis of C. glabrata fluconazole susceptibility. As it has been explained above for correct resistance determination the concentration of the drug in the well should be two times lower (32 µg/ml).

8 168 SZWEDA et al. In the case of amphotericin B, none of the tested strains examined by the Sensititre Yeast One test exhibited a MIC value above 2.0 µg/ml (CLSI recommended susceptibility breakpoint of 4 µg/ml), while in the ISYP test 26 were found to be resistant and were able to grow in the medium containing the antibiotic at a concentration of 2.0 µg/ml. Resistance to amphotericin B is rather rare among Candida spp., including C. glabrata, and thus the result of the ISYP seems to be unreliable. Results were nearly the same for 5-fluorocytosine in both tests, but discrepancies appeared in 4 cases. Yeasts of the genus Candida rarely exhibit primary resistance to this compound, thus only 3 strains were found to be resistant in the Sensititre Yeast One (CLSI recommended susceptibility breakpoint of 32 µg/ml) and 3 in the ISYP test, but only one isolate was found to be resistant in both tests. Forty-seven strains were resistant to itraconazole according to the Sensititre Yeast One, however for 30 of them the MIC value was exactly the same as a susceptibility breakpoint concentration according to CLSI (1.0 µg/ml). When MIC is equal to 1 µg/ml, the last well where growth is observed contains the agent at a concentration of 0.5 µg/ml. In the ISYP system nearly all isolates (except two) were able to grow in wells containing itraconazole at a concentration of 1 µg/ml. Taking this into account, the level of disagreement of results in this assay was about 74% (Table II). The level of disagreement of results in voriconazole susceptibility analysis was 33.8%. When the effect of fluconazole was compared, 15 strains were resistant (CLSI recommended susceptibility breakpoint 64 µg/ml) according to the Sensititre Yeast One as well as the microdilution method, whereas only 4 were identified as resistant according to the ISYP test. Interestingly, three strains classified as resistant in the ISYP were found sensitive in the Sensititre Yeast One and the CLSI recommended method, which means that only one strain was resistant according to all of the applied tests. This result could suggest some technical problems with plate preparation, which led to different final concentrations of the agent in each well, from those assumed by the manufacturer. Quick identification of a microorganism from infected patients and determination of the drug resistance of the yeast causing infection is extremely important in selecting an appropriate therapy. It seems to be especially significant when widespread resistance caused by excessive use of the most common antifungal drugs is observed. The ISYP system in its idea of species identification is similar to the API Candida test for Candida spp. In both of them the characteristic pattern of sugar assimilation is checked, then a specific code is calculated and the species name can be found in the codebook. From this point of view, the idea of the ISYP system is appropriate and should lead to correct results. However, as demonstrated

9 IDENTIFICATION AND SUSCEPTIBILITY TESTING FOR CANDIDA GLABRATA WITH ISYP 169 in our study, species identification is not always correct or even possible. In many cases, the achieved codes do not fit any of the codes from the codebook. In our opinion, the problem may occur due to the small number of reference and clinical strains obtained as assimilation patterns and used during the test designing procedure. In fact, the problems with correct identification of the tested strains were caused by two sugars: xylose and dulcitol. If these two substrates in the ISYP system were omitted, the result of the identification would be correct. Xylose is also included in the API Candida test, however, all strains tested were found xylanase negative in this assay. This could suggest that the reason for the observed differences was contamination of the xylose discs which are used in the ISYP system. In our opinion, the problem could be solved by replacement of the xylose disc with a dried substrate present in the well, as it is in the case of other sugars, or with a xylose derivative chromogenic substrate as it is in the API Candida test. This should eliminate the risk of contamination and generation of false positive results in this assay, which led to a wrong structure of the numerical code. Dulcitol is not included in the API Candida test. The observed differences of assimilation of this substrate could be very interesting for phenotypic differentiation of C. glabrata isolates, however, it should be additionally confirmed. If the strains really differ in their ability to assimilate this sugar, this should be taken into account in the code book. An undeniable advantage of the ISYP system is the possibility of identification and drug susceptibility testing using only one plate. This feature makes the system more useful than other tests dedicated only to identification or only for susceptibility testing. However, there is no possibility of determining the exact MIC value. Information if the strain is susceptible or resistant to particular agents is sufficient in clinical practice. At this point the idea of the test is good and would meet expectations. A good aspect of the test is also the possibility of simultaneous examination of ten compounds at the same time, although only two groups of antimycotics are analysed: polyenes and azoles. Results of the test can be interpreted in an easy way through simple visual observation of the wells colour change without using either a plate reader or any other expensive equipment. However, our studies showed there are still some problems with correct susceptibility establishment. Proper results for five tested agents were obtained only in 63% of cases compared to the Sensititre Yeast One, which is absolutely unsatisfactory. The problems with identification and susceptibility testing of Candida spp. isolates with the ISYP have been also widely discussed by Bicmen and co-workers [20]. In 2002 Morace and coworkers [21] evaluated the reliability of several commercially available tests among the other previous version of Integral System Yeast. The in-

10 170 SZWEDA et al. vestigation carried out clearly revealed that the test did not show a good agreement with NCCLS reference microdilution method and other commercially available tests. Apart from the above described matters in the ISYP system, some technical problems appeared. According to the instructions, 200 µl of suspension and two drops of vaseline oil should be added to each well. This volume seems to be too much because during movement of the plate the content of the well is sometimes spilt, and the results can be misleading. To summarize, we have no doubts that the idea of the ISYP system is certainly good. The combination of identification ability and drug susceptibility testing in one test is very important, especially from a clinical point of view. However, the current version of the ISYP has many disadvantages. We would like to encourage the manufacturer to make an effort and develop a new, more accurate version of the test. In terms of identification, a larger number of reference and clinical strains of yeasts from different species of the Candida genus should be tested. Moreover, according to the obtained results, all possible numerical codes corresponding to their species should be included in the code book. The mistakes in the determination of susceptibility probably come from loading the wrong amounts of agents to the wells, technical problems with immobilization/drying of the antimycotics in the plates, or some chemical modifications of the agents during drying, transport or storage of the plates. These problems might easily be eliminated. Of course in the new version of the ISYP, different values of fluconazole and other antifungal agents resistance MIC breakpoints for different species of Candida genus should be taken into account. For most agents at least two separated wells should be used. For example in the case of investigation of resistance of C. glabrata isolates the final concentration of fluconazole in the well should be 32 µg/ml. For analysis of resistance of other most common yeast pathogens belonging to the genus of Candida: C. albicans, C. tropicalis and C. parapsilosis the final concentration of fluconazole in the well should be 4 µg/ml. From the technical aspect, improved volume of wells and modifications of the amounts of some agents in wells need to be introduced, taking into account that the final concentration should be equal to half of its breakpoint MIC value. Additionally, including echinocandins in the test would be highly beneficial.

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