SHORT THESIS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY (Ph.D.) by Judit Szilágyi. Supervisor: László Majoros, M.D., Ph.D. UNIVERSITY OF DEBRECEN - PDF

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SHORT THESIS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY (Ph.D.) The in vitro and in vivo antifungal susceptibility testing of Candida parapsilosis sensu stricto, Candida orthopsilosis and Candida metapsilosis

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SHORT THESIS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY (Ph.D.) The in vitro and in vivo antifungal susceptibility testing of Candida parapsilosis sensu stricto, Candida orthopsilosis and Candida metapsilosis isolates by Judit Szilágyi Supervisor: László Majoros, M.D., Ph.D. UNIVERSITY OF DEBRECEN Doctoral School of Pharmaceutical Sciences Debrecen, 2012 The in vitro and in vivo antifungal susceptibility testing of Candida parapsilosis sensu stricto, Candida orthopsilosis and Candida metapsilosis isolates By Judit Szilágyi, M.Sc. Supervisor: László Majoros, M.D., Ph.D. Doctoral School of Pharmaceutical Sciences, University of Debrecen Head of the Examination Committee: Members of the Examination Committee: Árpád Tósaki, Ph.D., D.Sc. Anna Maráz, Ph.D., D.Sc. Miklós Vecsernyés, Ph.D. The Examination takes place at the Library of the Department of Pharmacology and Pharmacodynamics, Medical and Health Science Center, University of Debrecen. 11th of January 2013 at 11:00 Head of the Defense Committee: Reviewers: Members of the Defense Committee: Árpád Tósaki, Ph.D., D.Sc. Péter Kovács, M.D., C. Sc. Ilona Csányiné Dóczi, Ph.D. Árpád Tósaki, Ph.D., D.Sc. Ilona Csányiné Dóczi, Ph.D. Péter Kovács, M.D., C. Sc. Anna Maráz, Ph.D., D.Sc. Miklós Vecsernyés, Ph.D. The Ph.D. Defense takes place at the Lecture Hall of the 1 st Department of Internal Medicine, Medical and Health Science Center, University of Debrecen. 11th of January 2013 at 13:00 2 INTRODUCTION Despite the increasing number of antifungal agents the successful treatment of Candida infections remains a major challenge. This contradiction can be explained by the increasing frequency of the species, epidemiological alterations, poor clinical outcomes and the difficult diagnosis. During the last decades the invasive human infections caused by yeast species have become more frequent. The most frequent Candida species causing human infections are the C. albicans, C. glabrata, C. parapsilosis, C. tropicalis and C. krusei. Among these species the incidence of C. parapsilosis has risen the most since Due to the modern molecular biological studies C. orthopsilosis and C. metapsilosis have been separated from the C. parapsilosis sensu stricto less then ten years ago. Since then several studies have pointed out and still pointing out the clinical relevance of C. orthopsilosis and C. metapsilosis which account for approximately 1 % of the Candida invasive infections. The C. orthopsilosis and C. metapsilosis are phenotypically identical but genotypically different from the C. parapsilosis sensu stricto. In vitro studies showed that the antifungal susceptibility of C. orthopsilosis and C. metapsilosis are not identical with the C. parapsilosis sensu stricto s susceptibility. C. orthopsilosis and C. metapsilosis show relatively high minimum inhibitory concentrations (MICs) for fluconazole (FLU) but lower MICs for amphotericin B (AMB) compared to C. parapsilosis sensu stricto. Besides all of the three species have reduced in vitro echinocandin susceptibility. However echinocandin MICs for C. orthopsilosis and C. metapsilosis are lower compared to C. parapsilosis sensu stricto thus the newly separated two species are more susceptible against these antifungal agents. 3 Successful infection management can only be achieved by a properly applied and well-timed antifungal agent. As treatment guideline only exsists for the infections caused by the C. parapsilosis sensu stricto it is likely that new management strategy is needed to be established for the two newly separated species. The information about antifungal susceptibility of C. orthopsilosis and C. metapsilosis is scant in the literature which confirms the importance of our studies. Furthermore collecting data on echinocandin susceptibility of the less frequent Candida species is necessary as caspofungin (CAS) is increasingly used in treatments of invasive Candida infections. The verification of in vitro results by in vivo methods is essential. Although the in vitro results characterize a certain degree of antifungal activity it can differ from the actual effect at the site of infection due to the host-drug inetractions. Based on these thoughts we compared the in vitro activity of five antifungal agents (AMB, FLU, voriconazole (VOR), CAS, 5-fluorocytosine (5-FC)) against the Candida parapsilosis sensu stricto, C. orthopsilosis and C. metapsilosis species then the in vivo comparison was also performed with three out of five (AMB, FLU, CAS) selected drugs. Finally we wanted to know if the current treatment guideline of the C. parapsilosis sensu stricto infections could be applied for the C. orthopsilosis and C. metapsilosis species. 4 AIMS As in the current literature not too much information is available about the in vitro and mainly about the in vivo antifungal susceptibility of the recently separated Candida orthopsilosis and Candida metapsilosis species, furthermore therapeutic guidelines do not exist for the infections caused by these fungi we aimed in our study: to determine the minimal inhibitory concentrations of amphotericin- B, 5-fluorocytosine, fluconazole, voriconazole, posaconazole and caspofungin (in vitro microdilution method) against the Candida parapsilosis sensu stricto, Candida orthopsilosis and Candida metapsilosis species and in view of this information, to study and compare the in vitro pharmacodynamics of amphotericin-b, 5-fluorocytosine, fluconazole, voriconazole and posaconazole by time-kill curves (in vitro macrodilution method) within the psilosis group, furthermore to investigate and compare the in vivo activity of amphotericin-b, fluconazole and caspofungin against the Candida parapsilosis sensu stricto, Candida orthopsilosis and Candida metapsilosis species in neutropenic murine models, finally based on the results we wanted to determine the applicability of the Candida parapsilosis sensu stricto s international guideline for the therapies of infections caused by the Candida orthopsilosis and Candida metapsilosis species 5 MATERIALS AND METHODS Origin of the yeast isolates The six Candida parapsilosis sensu stricto, three Candida orthopsilosis and four Candida metapsilosis clinical isolates tested in our work were previously identified by molecular biological methods. The italian strains (C. parapsilosis sensu stricto CP120, CP117; C. orthopsilosis CP85, CP25, CP125; C. metapsilosis CP5, CP92, CP86) were provided by Arianna Tavanti (Universitá di Pisa, Pisa, Italy). Four in six C. parapsilosis sensu stricto (9150, 509, 2845, 896/1) and one in four C. metapsilosis (12821) isolates were identified in the Department of Medical Microbiology s Diagnostic Laboratory, University of Debrecen. In our experiments we also tested three ATCC reference strains (C. parapsilosis ATCC 22019, C. orthopsilosis ATCC 96139, C. metapsilosis ATCC 96144). In vitro antifungal susceptibility testing Determination of the minimal inhibitory concentracion The minimal inhibitory concentrations (MIC) of AMB, 5-FC, FLU, VOR, POS and CAS were performed minimum two times in accordance with the CLSI s (Clinical and Laboratory Standards Institute) M27-A3 document. The FLU, 5- FC and CAS antifungal agents were dissolved in sterile distilled water while the other drugs were dissolved in 100 % DMSO. AMB, VOR, POS and CAS were tested at 0,015-8 µg/ml, 5-FC and FLU were tested at 0,12-64 µg/ml concentrations. For the fungi suspensions prepared in 0,85 % saline with 0,5 McFarland density we used 24 hours old colonies, cultured on Sabouraud agar plate. We used RPMI-1640 for the appropriate cell count adjustment which was 6 10 4 CFU/mL in case of the AMB and 10 3 CFU/mL in case of the other tested drugs. The 96-well Elisa-plates used for the MIC determination contained fungi control (antifungal drug free) and media control (yeast free) wells. After 48 (AMB, 5- FC, FLU, VOR, POS) and 24 (CAS) hours of incubation at 35 C the contents of the wells were suspended with pipette and the results were read visually. Data evaluation The MIC of the AMB is the lowest concentration of the antifungal drug which does not cause visible growth (total inhibition) compared to the control while the MIC of the other antifungal agents cause 50 % decrease (prominent inhibition) compared to the control. E-test As E-test is considered the best method for amphotericin B resistance detection in Candida strains, amphotericin B MICs were also determined by this method. E-test was carried out using 0,5 McFarland density fungal suspensions prepared with 24 hours old cultures in 0,85 % saline and RPMI-1640 agar supplemented with 2 % glucose. These suspensions were evenly spreaded on the surface of the RPMI agar plates with steril swabs, after the plates were dried we placed the test stripes impregnated with AMB onto them. Results were read visually after 24 and 48 hours of incubation at 35 ºC. Time-kill curves Time-kill studies were performed according to the standardised method of Klepser and his colleagues. We prepared 10 5 CFU/mL starting inoculum from each tested isolates in RPMI-1640 liquid media using densitometer. Antimycotics (5-FC, FLU, VOR, POS) were tested at 0,5-16 MIC 7 concentrations. As the maximal attainable free amphotericin B concentration in the serum is less than 1 µg/ml, the highest tested amphotericin B concentration was only 4 µg/ml, regardless of the actual MIC. Test tubes containing the media, the fungi suspension and the antifungal agents at different concentrations were incubated for 48 hours with agitation in the dark at 35 C. At 0, 4, 8, 12, 24 and 48 hours µl samples were removed from the tubes and serially diluted 10-fold in sterile saline; four 30-µL aliquots were subsequently plated onto Sabouraud agar plates. In those cases when colony counts were suspected to be less than 1000 CFU/mL undiluted samples were plated as above. The limit of quantification is 50 CFU/mL. Agar plates were incubated after the inoculated liquid was dried for minutes at room temperature, 48 hours later we counted the grown colonies and determined the CFU (colony forming unit) according to the degree of the dilutions. Finally the CFU results versus time were plotted. All of the time-kill experiments were performed minimum twice and the results were averaged. Time-kill curves were prepared using the computer curve-fitting software (GraphPad Prism 4.03 for Windows). Fungicidal activity was defined as a 99,9% ( 3 log 10 ) reduction in viable CFU/mL of the starting inoculum. Fungistatic activity was defined as detectable colony number decrease, which do not reach 99,9% ( 3 log 10 ) compared to the starting inoculum. In vivo antifungal susceptibility testing Animals In our experiments we used female BALB/c mice weighing g which were maintained in accordance with the Guidelines for the Care and Use of Laboratory Animals. Each group consisted of 7-11 animals. The number of the in vivo experiment s permission: 12/2008 DE MÁB. 8 Immunosuppression of the BALB/c mice Mice were immunosuppressed using a single 200 mg/kg intraperitoneal cyclophosphamide dose three days prior to infection. In order to prevent emerging Gram-negative bacterial infections all murine recieved 5 mg/kg subcutan ceftazidime from the day of the infection until the end of the experiment. Preparation of the infectious doses For the infective doses preparation we plated the isolates of the three species (C. parapsilosis sensu stricto 9150, 896/1, C. orthopsilosis CP25, CP125, CP85, C. metapsilosis CP5, CP92, CP86) onto Sabouraud agar plates on two consecutive days and then the renewed strains were plated onto three or four Sabouraud agar plates again. The grown isolates were taken from the surface of the agar plates with sterile swab and suspended in sterile saline. These suspensions were centrifugated for four times/10 minutes at 30000g. We removed the supernatant from the cells after each centrifugation and added ml fresh, sterile saline again to the them. After the last centrifugation we removed the supernatant again and added 3 ml of sterile saline to the fungi cells. From this cell suspension we prepared a 10-fold dilution in two steps and adjusted the required cell count of the infective dose with Burker chamber. The punctuality of the infective dose s cell count was checked by quantitative inoculation. Infection of mice Mice were infected with the fungi suspension intravenously (0,2 ml/mouse) through the lateral tail vein on the fourth day of the immunosuppression. The infective dose of the C. metapsilosis isolates was CFU/mouse, and slightly lower, CFU/mouse in the case of the C. orthopsilosis and C. 9 parapsilosis sensu stricto strains. These doses did not cause lethality in the control groups. Antifungal therapy The intraperitoneal AMB (1 mg/kg/day), FLU (1, 5, 10, 20 mg/kg/day) and CAS (1, 2, 5 mg/kg/day) treatments of the mice were started 24 hours after infection and lasted for five days. All drug doses were given in a 0,5 ml bolus. In the experiments we applied the currently suggested doses for the human therapies. The control groups were given 0,5 ml sterile saline. Organ culture For the quantitative CFU determination mice still alive on the 7 th day were dissected after cervical dislocation, the kidney pairs were removed and homogenised aseptically. After one ml sterile saline was added to the homogenate an aliquot was serially diluted 10-fold. Aliquots of 100 µl of the appropriate dilutions were plated on Sabouraud agar plates. The numbers of the colonies grown on the agar plates were determined after 48 hours of dark incubation at 35 C. For the statistical analysis we used the Kruskal-Wallis test in case of the fungi cultured on the agar from the kidneys. P values of 0,05 were regarded as significant. 10 RESULTS Results of the in vitro antifungal susceptibility testing Minimal inhibitory concentrations MIC values of the ATCC quality control strains were in the acceptable CLSI ranges. The amphotericin B MICs obtained with the CLSI and Etest methods agreed within ±1 dilution and ranged between 0,25-2 µg/ml. In the case of the E-test method 1 µg/ml was the highest read concentration. Using the CLSI method the C. orthopsilosis CP25 isolate was the most susceptible against this polyen antifungal agent while using the E-test the C. metapsilosis ATCC and CP86 strains proved to be the most susceptible. Against the triazole antifungal agents all of the tested isolates were susceptible. In the case of FLU the C. parapsilosis sensu stricto isolates showed greater susceptibility (MIC: 0,5-2 µg/ml) than the C. orthopsilosis and C. metapsilosis species (the highest MIC: 8 µg/ml). Applying the 24 hours incubation the MICs of the isolates were 1-4 dilutions lower than in the case of the 48 hours incubation. We observed 0,015-0,12 µg/ml MIC ranges for VOR and POS. The C. parapsilosis sensu stricto 2845 and CP120 strains were the most susceptible to VOR (0,015 µg/ml). The C. metapsilosis CP5 (0,015 µg/ml) and CP92 (0,015 µg/ml) were the most susceptible strains to POS. The 5-FC MICs were 0,12 µg/ml in case of all of the tested isolates. After 24 hours incubation the CAS MIC values of the two newly separated C. orthopsilosis and C. metapsilosis species were lower (0,12-1 µg/ml) than the MICs of the C. parapsilosis sensu stricto isolates (0,5-2 µg/ml). 11 Results of the time-kill studies Amphotericin B In the time kill studies, AMB was fungicidal at 1 to 2 µg/ml (0,5 2 MIC) after 24 and 48 h against the C. parapsilosis sensu stricto ATCC strain. However against the 896/1 and also against the CP120 isolates slightly higher 1 to 4 (1 4 MIC) and 2 to 4 (2 4 MIC) µg/ml AMB concentrations were fungicidal, respectively. The remaining C. parapsilosis sensu stricto clinical isolates (9150, 509, 2845, CP117) were also killed at 4 µg/ml AMB after 48 h ( 99,9 % CFU reduction). After 24 h of incubation AMB at 2-4 (2-16 MIC) µg/ml concentrations was fungicid against all of the tested C. orthopsilosis strains. The CP125 isolate was most readily killed by AMB; killing was observed even at 1 µg/ml (1 MIC) AMB concentration after 24 h. After 48 h, 1 to 4 µg/ml of AMB were fungicidal ( 99,9 % CFU decrease) against 3 of 5 C. metapsilosis isolates (CP92, CP86 and 12821). In the case of the remaining CP5 and C. metapsilosis ATCC strains we observed fungicidal killing at slightly higher, 2 to 4 µg/ml (2 4 MIC) AMB concentrations also after 48 h. Against the C. metapsilosis CP86 isolate AMB was fugicid even after 12 h at 4-8 MIC concentrations. 5-fluorocytosine 5-FC caused less than 99,9 % reduction in viable CFU/mL of the starting inoculum, thus proved to be fungistatic against all of the members of the psilosis group. None of the tested 5-FC concentrations were fungicidal against the C. parapsilosis ATCC isolate. Some, but not all C. orthopsilosis (CP85, CP125) and C. metapsilosis (CP92, 12821) isolates required higher 2 4 MIC (0,24-0,48 µg/ml) 5-FC concentrations for greater effective inhibition 12 ( 99,9 % CFU decrease). Similarly to the other members of the psilosis group we did not observe the 5-FC s fungicidal effect against the C. metapsilosis isolate. Fluconazole Triazoles were fungistatic against all three Candida spp. (less than 99,9 % reduction in viable CFU/mL of the starting inoculum). FLU was fungistatic even at 1 MIC against all C. parapsilosis sensu stricto isolates. The lower 0,5 MIC FLU concentration more moderately inhibited the growth of the CP120 C. parapsilosis sensu stricto isolate than the higher 8-16 MIC values. C. orthopsilosis CP25 was inhibited at 1 MIC (8 µg/ml). C. orthopsilosis CP85, CP125 and ATCC were fungistatically inhibited at higher, 2-16 times FLU MICs ( µg/ml). The majority of the C. metapsilosis strains (ATCC 96144, CP5, CP86, 12821) were inhibited fungistatically at 1 MIC (4 µg/ml), whereas isolate CP92 was only inhibited by higher 2 MIC (16 µg/ml) FLU. Voriconazole While testing the C. parapsilosis sensu stricto isolates VOR caused growth inhibition ( 99,9 % CFU reduction) at low concentrations 1 MICs (0,015-0,12 µg/ml). The C. orthopsilosis ATCC 96139, CP85 and CP125 isolates were inhibited at 0,5 µg/ml or only higher (4 8 MIC values) VOR while isolate CP25 was effectively inhibited at lower, 1 MIC (0,12 µg/ml). While C. metapsilosis ATCC strain was inhibited even by 2 MIC (0,25 µg/ml), all C. metapsilosis clinical isolates were inhibited at VOR concentrations of 4-8 MICs (0,25-0,5 µg/ml) and no significant fungistatic inhibition was observed at concentrations that were twice the MICs. 13 Posaconazole POS caused growth inhibition ( 99,9 % CFU reduction) in the case of the C. orthopsilosis and C. metapsilosis ATCC strains and all clinical isolates at 0,03 0,06 and 0,015 0,03 µg/ml concentrations, respectively. At 0,25-0,5 µg/ml (4-8 MIC) POS concentrations more than 1 lo g10 colony number decreas was detected for the CP85 and CP25 C. orthopsilosis and the CP92 C. metapsilosis clinical isolates (1,2 1,48 log 10 and 2,22 log 10 CFU/mL, respectively). We did not test the C. parapsilosis isolates against POS, and all of the other members of the group against CAS as our team has published these results previously. Results of the in vivo antifungal susceptibility testing C. parapsilosis sensu stricto isolates While 1 mg/kg/day FLU was ineffective (P 0,05) against the C. parapsilosis sensu stricto 9150 strain in neutropenic mice 5, 10 (P 0,05) and 20 mg/kg (P 0,01) FLU daily doses significantly reduced the tissue fungal burden. CAS was only effective at 5 mg/kg daily dose (P 0,05). AMB at 1 mg/kg daily dose also significantly reduced the tissue fungal burden (P 0,001). Similarly to the 9150 isolate all of the tested FLU doses (5 mg/kg-p 0,05; 10 mg/kg-p 0,01; 20 mg/kg-p 0,001) proved to be effective against the 896/1 strain except of the 1 mg/kg dose. Against this isolate 2 mg/kg daily CAS was also effective (P 0,05) in addition to th
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