Prace oryginalne / Original papers Mikologia Lekarska 2007, 14 (1): 23-29

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1 Prace oryginalne / Original papers Mikologia Lekarska 2007, 14 (1): Copyright 2006 Cornetis ISSN X Characteristics of phenotypic species and intraspecies features of fungal strains isolated from patients with renal failure undergoing chronic haemodialysis Charakterystyka cech fenotypowych gatunkowych i wewn¹trzgatunkowych szczepów grzybów wyizolowanych od pacjentów z niewydolnoœci¹ nerek poddanych hemodializom Piotr Kurnatowski 1, Micha³ Nowicki 2, Ilona Kurnatowska 2, Agata Drozdowska 1 1 Department of Biology and Medical Parasitology, Chair of Biology and Medical Genetics, Medical University of Lodz, Poland 2 Department of Nephrology, Hypertension and Kidney Transplantation, Medical University of Lodz, Poland Abstract Streszczenie Introduction: Patients with renal failure are more frequently at risk of fungal infections than healthy individuals. An increased susceptibility to infections is the result of coexisting immunity disorders, both humoral and cellular but the most common risk factors in renal failure continue to be the invasive diagnostic/therapeutic procedures and the use of broad-spectrum antibacterial agents for frequent bacterial infections. The aim of the study: The aim of the study was to evaluate the prevalence of fungi in patients with end-stage renal failure undergoing haemodialysis and to describe selected species and intraspecies phenotypic features of isolated fungal strains. Material and methods: The study group comprised 56 patients from a single dialysis unit (35 men and 21 women aged from 25 to 78 years; mean age x =55.4±3.75 years) dialysed for the mean period of 36.2 (±1.62) months. All patients were on chronic haemodialysis therapy (4 hours sessions, 3 times per week). Material for mycological examinations included washings from the oral cavity and samples of urine and faeces. In order to evaluate morphological and biochemical features of fungi the following methods were applied: 1. macrocultures in solid Sabouraud medium, 2. direct microscopic slides, 3. API 20 C AUX test and 4. API ZYM test (biomérieux). Results: A hundred and forty-five samples for the mycological examinations were collected: 56 samples from the oral cavity washings, 56 from faeces and 33 from urine. Totally, 80 fungal strains were isolated from those samples: 48 strains from the oral cavity and 28 and 4 strains from faeces and urine samples, respectively. Frequency of isolation of fungi from patients with renal failure undergoing long-term dialysis was evaluated at the level of 85.7 % for the samples collected from oral cavity washings and 50.0% and 7.1 % for faeces and urine samples, respectively. Of all isolated strains 76 belonged to the genus Candida, 2 to the genus Saccharomyces and the remaining 2 to the genera: Cryptococcus and Geotrichum. Candida albicans was the most frequently isolated pathogen (58.8±5.50%). The highest mean enzymatic activity of hydrolases of the isolated fungal strains showed: e2 alkaline phosphatase, e6 leucine arylamidase, e11 acid phosphatase and e12 naphthol-as-bl-phosphohydrolase, whereas the lowest were: e7 valine arylamidase, e13 -galactosidase, e14 -galactosidase, e16 -glucosidase, e17 -glucosidase, e18 N-acethyl- -glucosylamidase and e19 -mannosidase. The majority of C. albicans strains isolated from biological materials from haemodialysed patients were classified to biotype D 3 (acc. to Kurnatowski et al.) 46.8±7.28%, and to biotype E (acc. to Williamson or C6 acc. to Kurnatowski et al.) 23.4±6.18%. Conclusions: Fungi were isolated in almost all chronic haemodialysis patients with Candida albicans predominating among all strains. The majority of strains isolated from oral cavity, faeces and urine of our patients showed the highest and the lowest mean enzymatic activity of the same hydrolases like described in case of immunocompromised patients after renal transplantation, hospitalised in intensive care units, with cancer or other chronic diseases. Key words: renal failure, haemodialysis, fungi Wprowadzenie: U pacjentów z niewydolnoœci¹ nerek, czêœciej ni w populacji ludzi zdrowych, dochodzi do zara eñ grzybami. Zwiêkszona podatnoœæ na infekcje jest skutkiem towarzysz¹cych zaburzeñ odpornoœci, zarówno humoralnej, jak i komórkowej, lecz najczêstsze czynniki ryzyka w niewydolnoœci nerek wci¹ stanowi¹ inwazyjne metody diagnostyczno-lecznicze i stosowanie œrodków przeciwbakteryjnych o szerokim spektrum dzia³ania z powodu czêstych infekcji bakteryjnych. Cel pracy: Celem pracy by³a ocena prewalencji grzybów u pacjentów ze schy³kow¹ niewydolnoœci¹ nerek, leczonych hemodializami oraz okreœlenie wybranych cech fenotypowych gatunkowych i wewn¹trzgatunkowych wyizolowanych szczepów grzybów. Materia³ i metody: Badaniem objêto 56 pacjentów (35 mê czyzn i 21 kobiet w wieku lat; œrednia wieku 55,4±3,75 roku) dializowanych przewlekle przez œredni okres 36,2±1,62 miesi¹ca. Wszyscy byli leczeni przewlek³¹ hemodializ¹ (4-godzinne sesje, 3 razy w tygodniu). Materia³ do badañ stanowi³y pop³uczyny treœci jamy ustnej, mocz i ka³. W celu oceny cech morfologicznych i biochemicznych grzybów u yto nastêpuj¹ce metody: 1) makrohodowle na pod³o u sta³ym Sabourauda, 2) preparaty bezpoœrednie do oceny mikroskopowej, 3) test API 20 C AUX, 4) test API ZYM (biomérieux). Wyniki: Ogó³em pobrano do badañ 145 prób: 56 pop³uczyn jamy ustnej, 56 ka³u oraz 33 moczu. ¹cznie wyizolowano 80 szczepów grzybów: 48 szczepów z jamy ustnej i odpowiednio 28 i 4 szczepy z ka³u i moczu. Czêstoœæ izolowania grzybów od pacjentów z niewydolnoœci¹ nerek leczonych przewlek³ymi dializami wynios³a 85,7% dla próbek pobranych z jamy ustnej oraz odpowiednio 50,0% i 7,1% dla próbek ka³u i moczu. Spoœród wyizolowanych szczepów 76 nale a³o do rodzaju Candida, 2 do rodzaju Saccharomyces oraz po jednym szczepie do rodzajów Cryptococcus i Geotrichum. Najczêœciej oznaczanym gatunkiem by³a Candida albicans (58,8±5,50%). Najwy sz¹ œredni¹ aktywnoœæ enzymatyczn¹ szczepów grzybów wyizolowanych z poszczególnych ontocenoz wykaza³y hydrolazy: e2 fosfataza zasadowa, e6 arylamidaza leucynowa, e11 fosfataza kwaœna i e12 naftolu-as-bl-fosfohydrolaza; najni sz¹ zaœ: e7 arylamidaza walinowa, e13 -galaktozydaza, e14 -galaktozydaza, e16 -glukozydaza, e17 -glukozydaza, e18 N-acetylo- -glukozyloamidaza i e19 -mannozydaza. Wiêkszoœæ szczepów C. albicans wyizolowanych z materia³ów biologicznych pobranych od pacjentów hemodializowanych zaklasyfikowano do biotypu D 3 (wg Kurnatowskiego i wsp.) 46,8±7,28%, oraz do biotypu E (wg Williamsona lub C 6 wg Kurnatowskiego i wsp.) 23,4±6,18%. Wnioski: Grzyby by³y izolowane u prawie wszystkich pacjentów przewlekle hemodializowanych, a Candida albicans by³a dominuj¹cym gatunkiem wœród wszystkich oznaczonych szczepów. Wiêkszoœæ szczepów wyizolowanych z jamy ustnej, ka³u i moczu badanych pacjentów, wykaza³o najwy sz¹ i najni sz¹ œredni¹ aktywnoœæ enzymatyczn¹ takich samych enzymów hydrolitycznych jak opisywano w przypadkach pacjentów z obni on¹ odpornoœci¹, po przeszczepach nerek, hospitalizowanych w oddzia³ach intensywnej opieki medycznej, pacjentów z rakiem czy cierpi¹cych z powodu innych przewlek³ych chorób. S³owa kluczowe: niewydolnoœæ nerek, hemodializy, grzyby Address for correspondence: Prof. Piotr Kurnatowski, M.D., Department of Biology and Medical Parasitology Medical University of Lodz, Haller s Square 1, Lodz, Poland, pkurnatowski@yahoo.com 23

2 Characteristics of phenotypic species and intraspecies features of fungal strains isolated from patients with renal failure undergoing... Mikologia Lekarska 2007, 14 (1) 24 Introduction Patients with renal failure are at much higher risk of fungal infections than healthy individuals. An increased susceptibility to infections is the result of coexisting immunity disorders, both humoral and cellular (1). Fungal infections often develop quite fast, frequently without serious general symptoms, which may be the result of decreased immune response during uremia. The additional factors responsible for the development of mycoses include: frequent hospitalisations, blood transfusions and other procedures, such as intraperitoneal catheters placement for peritoneal dialysis and intravenous for haemodialysis, the presence of arteriovenous fistulae or artificial vascular prostheses (2). A higher risk of fungal infections in patients with renal failure is also a result of frequent use of broad-spectrum antibacterial antibiotics and long term treatment with corticosteroides, immunosuppressive and cytostatic drugs (1, 2). The risk also increases with invasive methods of diagnostics and treatment used in the predialysis period. For example lithotripsy by means of extracorporal generated shock wave or noninvasive method of renal stones destruction can be the cause of fungemia, or even endocarditis or endophthalmitis in patients who had coexisting infection or colonisation of the urinary tract by Candida sp. (3, 4). The number of haemodialysed patients with diabetes as the initial cause of end-stage renal failure is also increasing. Diabetes is one of the most important risk factors predisposing to the development of mycoses (5, 6). The available literature concerning fungal infections in patients with end-stage renal disease has focused on renal transplant recipients or on fungal peritonitis in patients on peritoneal dialysis. Peritoneal dialysis is an alternative for haemodialysis. In such case fungal peritonitis is connected with higher morbidity and mortality rates in comparison with bacterial peritonitis (7). Infection can be transmitted after contamination of a catheter, or a site of a needle insertion, or through internal passage during intestinal perforation, or after crossing peritoneal barrier or through the vagino-peritoneal fistulae. The most important risk factor responsible for the development of fungal peritonitis in ambulatory patients undergoing peritoneal dialysis at an outpatients clinic is administration of antibacterial drugs because of bacterial peritonitis. The most common etiological factor of fungal infections in case of ambulatory peritoneal dialysis are species from genus Candida, such as: C. albicans, C. tropicalis and C. parapsilosis (8-11). Despite the fact that there has been a great progress in the management of end-stage renal failure with organ transplantation and the long-term success of this procedure is high, approximately 5% of patients develop a fungal infection after transplantation which carries a high morbidity and mortality rates. Fungal complications after renal transplantation can be divided into two groups. The first group of infections occurs during the first four weeks when nosocomial infections caused by species from genus Candida are observed, the same as in other patients suffering from critical conditions after surgical procedures. The second group of infections occurs after four weeks from the surgery, when cumulated effects of immunosuppressive drugs create favourable conditions for the development of opportunistic pathogens, such as Cryptococcus neoformans or different species from Aspergillus and Penicillium genera (12). Investigations concerning prevalence of fungal infections among patients after renal transplantation conducted in our centre revealed frequent occurrence of such infections in this particular group of patients; most frequently multifocal infections of the oral cavity and anus ontocenoses were observed (13). The most common pathogen isolated from patients who were at least three months after renal transplantation was C. albicans, whereas strains of C. glabrata, C. guilliermondii, C. krusei and Saccharomyces cerevisiae were found rarely (14). Infection of the donor s kidney can also be the cause of mycoses caused by: C. albicans, Cryptococcus neoformans, Histoplasma capsulatum and Scedosporium apiospermum after transplantation (15, 16). The aim of the study was to evaluate the prevalence of fungi in patients with end-stage renal failure undergoing chronic haemodialysis and to describe selected species and intraspecies phenotypic features of isolated fungal strains. Material and methods The study group comprised 56 patients (35 men and 21 women aged years; mean age 55.4±3.75 years) undergoing chronic dialysis for the period over three years (x = 36.2±1.62 months). Causes of end-stage renal failure were as following: chronic glomerulonephritis (50.0± 6.68%), polycystic kidney disease (12.5±4.42%), interstitial nephropathy (12.5±4.42%), diabetic nephropathy (8.93±3.81%), nephrosclerosis (5.36±3.01%), unknown reasons (10.7±4.13%). All patients were dialysed three times a week for 4-5 hours. Thirtythree patients presented residual diuresis above 100 ml. At the moment of collection of material for mycological examinations none of the patients revealed clinical symptoms of viral or bacterial infection, or presented complaints which might suggest fungal infection or was treated with antibiotics over four previous weeks. One patient was treated long-term with prednisolone because of coexisting systemic vasculitis. Fifty-two patients were dialysed through fixed vascular access arteriovenous fistulae formed on their own vessels, 2 patients through fistulae formed with the use of vascular prosthesis and the remaining 2 patients were dialysed through the permanent catheter. None of the patients had an inserted ureteral catheter. Biological material used for the mycological examinations included washings from the oral cavity, and samples of urine and faeces. Collection of material was preceded by history taking and physical examination of patients. In order to evaluate morphological and biochemical features of fungi the following tests were used: 1) macrocultures in solid Sabouraud medium, 2) direct microscopic slides, 3) API 20 C AUX test (biomérieux), 4) API ZYM test (biomérieux). Collected material from different ontocenoses was cultured into solid-agar Sabouraud medium, incubated in temperature +37ºC for 24 hours and then left in room temperature +20 C for hours. After macroscopic evaluation of obtained cultures including: colour, shape, polish, margins of cultures, structure of surface and relation towards the surface of agar, the microscopic analyses of direct preparations in 0.9% NaCl were performed. After those examinations fungal strains were subjected to API 20 C AUX and API ZYM tests (biomérieux, France). API 20 C AUX test is the biochemical identification series based on the evaluation of fungal ability to assimilate carbon (auxanogram) from 19 compounds and control, i.e.: GLU (glucose), GLY (glycerol), 2KG (2-keto-D-gluconate), ARA (L-arabinose), XYL (D-xylose), ADO (adonitol), XLT (xylitol), GAL (galactose), INO (inositol), SOR (sorbitol), MDG (L-methyl-D-glucoside), NAG (N-acetyl-glucosamine), CEL (cellobiose), LAC (lactose), MAL (maltose), SAC (saccharose), TRE (trehalose), MLZ (melesitose), RAF (rafino-

3 Charakterystyka cech fenotypowych gatunkowych i wewn¹trzgatunkowych szczepów grzybów wyizolowanych od pacjentów z niewydolnoœci¹ nerek poddanych hemodializom se), 0 (control). The way of execution of API 20 C AUX test is described by the manufacturer. The final determination of fungal species is made using the rule of number identification included in the Code Book (Analytical Profile Index, biomérieux). API ZYM test (biomérieux) is a semiquantitative method in order to determine the hydrolytic activity. The strips of API ZYM consist of microtubes which contain substrates for identification and activity evaluation of 19 enzymes. The way of preparing fungal strains, performing the test and reading the results was made according to the manufacturer s instruction. Activities of enzymes were determined in nanomoles of hydrolysed substrate according to the intensity of colour reaction in six-grade scale from 0 to 5 scores as following: 0=0 nanomoles (lack of reaction), 1=5 nanomoles, 2=10 nanomoles, 3=20 nanomoles, 4=30 nanomoles, 5 40 nanomoles. Results A total of 145 samples for the mycological examinations were collected: 56 samples of washings from the oral cavity, 56 samples of faeces and 33 of urine, out of which 80 fungal strains were in all isolated: 48 strains from the oral cavity; 28 and 4 strains from faeces and urine samples, respectively. Of 80 isolated strains 76 belonged to genus Candida, 2 to genus Saccharomyces and the remaining 2 strains to genera Cryptococcus and Geotrichum (tab. I). From the oral cavity and faeces Candida albicans strains were the most frequently isolated, 34 and 13 strains respectively. This species was not detected in examined urine samples. In 8 cases C. albicans cells were found in two ontocenoses (oral cavity and gastrointestinal tract) of the same patient. From faeces of 6 patients C. humicola strains were isolated, but from oral cavity washings only one strain of this species; also one from urine samples. The fungi belonged to C. glabrata species were detected from 5 of examined patients faeces and from 2 of oral cavity washings; C. tropicalis from 3 oral cavity samples and from 1 urine sample. Other fungal strains were isolated from only single patients. C. albicans strains isolated from the oral cavity revealed the activity of 13 from 19 hydrolases of the API ZYM test. The highest enzymatic activity showed the following hydrolases: e11 acid phosphatase, e2 alkaline phosphatase, e6 leucine arylamidase and e12 naphthol-as-bl-phosphohydrolase; Table I: Prevalence of fungal species in the examined ontocenoses Tabela I: Prewalencja gatunków grzybów w badanych ontocenozach Species Gatunek Prevalence of fungal species Prewalencja gatunków grzybów oral cavity jama ustna n=56 faeces ka³ n=56 urine mocz n=33 1. Candida albicans 60.7% 23.2% 0 2. Candida glabrata 3.57% 8.93% 0 3. Candida guilliermondii % 4. Candida humicola 1.78% 10.7% 3.03% 5. Candida kefyr 1.78% Candida krusei % 0 7. Candida lambica 1.78% 1.78% 0 8. Candida parapsilosis 1.78% 1.78% 0 9. Candida rugosa % 10. Candida zeylanoides 1.78% Candida tropicalis 5.36% % 12. Cryptococcus albidus 1.78% Geotrichum penicillatum 1.78% Saccharomyces cerevisiae 1.78% 1.78% 0 whereas the lowest activity was observed in case of: e5 lipase (C14), e13 -galactosidase, e16 -glucosidase, e18 N-acethyl- -glucosylamidase. Lack of enzymatic activity was observed for the enzymes: e9 trypsin, e10 -chymotrypsin, e14 -galactosidase, e15 -glucuronidase, e17 -glucosidase and e20 -fucosidase (tab. II, III). Strains of C. albicans isolated from faeces revealed the highest enzymatic activity of the same hydrolases. No enzymatic activity was seen in case of 7 hydrolases: e5 lipase (C14), e9 trypsin, e10 -chymotrypsin, e13 -galactosidase, e14 -galactosidase, e15 -glucuronidase and e20 -fucosidase (tab. II, IV). Statistical analysis of hydrolytic activity revealed that the highest mean activity values of enzymes secreted by strains obtained from oral cavity and faeces did not show statistically significant differences. Such differences were found in case of enzymes: e4 esterase lipase (C 8), e7 valine arylamidase, e19 -mannosidase. Esterase lipase and valine arylamidase Table II: Hydrolytic activity of C. albicans strains isolated from the oral cavity and faeces Tabela II: Aktywnoœæ enzymatyczna szczepów C. albicans wyizolowanych z jamy ustnej i ka³u Enzyme Enzym Mean activity±s [nmoles of hydrolysed substrate] Œrednia aktywnoœæ±s [nmole rozk³adanego substratu] oral cavity jama ustna [n=34] faeces ka³ [n=13] Statistical level of difference Statystyczna istotnoœæ ró nicy p e2 Phosphatase alkaline 25.0± ±3.37 p>0.5 Fosfataza zasadowa e3 Esterase (C 4) 9.26± ±3.13 p>0.3 Esteraza e4 Esterase lipase 4.41± ± <p<0.01 (C 8) / Lipaza esterazowa e5 Lipase (C 14) / Lipaza 0.735± p>0.5 e6 Leucine arylamidase 19.26± ±2.85 p>0.3 Arylamidaza leucynowa e7 Valine arylamidase 1.76± <p<0.05 Arylamidaza walinowa 3.46±0.666 e8 Cystine arylamidase 1.18±0.369 p>0.6 Arylamidaza cystynowa 1.54±0.666 e9 Trypsin / Trypsyna e10 Chymotrypsin Chemotrypsyna e11 Phosphatase acid 37.7± ±2.08 p>0.7 Fosfataza kwaœna e12 Naphthol-AS-BL- 13.7± ±1.74 p>0.5 -phosphohydrolase Fosfohydrolaza naftolowa-as-bl e13 -galactosidase 0.588± p>0.5 -galaktozydaza e14 -galactosidase -galaktozydaza e15 -glucuronidase -glukuronidaza e16 -glucosidase 0.735±0.308 p>0.9 -glukozydaza 0.769±0.521 e17 -glucosidase ± 1.54 p>0.1 -glukozydaza e18 N-acetyl ±0.352 p>0.6 -glucosylamidase N-acetyl- - -glukozylaminidaza 0.769±0.521 e19 -mannosidase 3.82± <p<0.05 -mannozydaza 2.31±0.720 e20 -fucosidase -fukozydaza 25

4 Characteristics of phenotypic species and intraspecies features of fungal strains isolated from patients with renal failure undergoing... Mikologia Lekarska 2007, 14 (1) Table III: Percentage of C. albicans strains (n=34) isolated from the oral cavity with enzymatic activity of hydrolases Tabela III: Odsetek szczepów C. albicans (n=34) wyizolowanych z jamy ustnej wykazuj¹cych aktywnoœæ enzymów hydrolitycznych e ± ± ±8.33 e3 2.94± ± ± e4 11.8± ± e5 94.1± ± ± e6 11.8± ± ± ±7.27 e7 64.7± ± e8 76.5± ± e ± ± ±4.86 e ± ± ±4.03 e ± ± e ± ± e e ± ± e ± ± Table IV: Percentage of C. albicans strains (n=13) isolated from faeces with enzymatic activity of hydrolases Tabela IV: Odsetek szczepów C. albicans (n=13) wyizolowanych z ka³u wykazuj¹cych aktywnoœæ enzymów hydrolitycznych e2 7.69± ± ± ±11.7 e3 7.69± ± ± ±7.39 e ± ± e e ± ± ±11.7 e7 30.8± ± e8 69.2± ± e ± ±10.0 e ± ± e e ± ± e ± ± e ± ± e ± ± showed significantly higher activity in strains from faeces; -mannosidase in strains from oral cavity (tab. II). Other strains from genus Candida: C. glabrata, C. humicola, C. kefyr, C. lambica, C. parapsilosis, C. zeylanoides, isolated from the oral cavity showed the activity of 6 from 19 API ZYM hydrolases; always the highest for e11 acid phosphatase, and also high for e2 alkaline phosphatase and e6 leucine arylamidase. Fungi from genera Cryptococcus, Geotrichum, Saccharomyces revealed 6 8 hydrolases with similar activity. Only C. tropicalis strains distinguished for their 9 11 hydrolases activity which did not occur in the above mentioned strains: e16 -glucosidase, e17 -glucosidase, e18 N-acethyl- - -glucosylamidase and e19 -mannosidase. The Candida strains obtained from faeces, except C. albicans, released 7 11 enzymes towards API ZYM substrates; always lack of: e5 lipase (C14), e9 trypsin, e10 -chymotrypsin, e13 -galactosidase, e14 -galactosidase, e15 -glucuronidase and e20 -fucosidase was observed (tab. V-VII). Strains isolated from urine: C. guilliermondii, C. humicola, C. rugosa, C. tropicalis showed the highest enzymatic activity of hydrolases: e11 acid phosphatase, e2 alkaline phosphatase, e6 leucine arylamidase and e12 naphthol-as-bl-phosphohydrolase, and the lowest activity was characteristic for enzymes: e7 valine arylamidase, e18 N-acethyl- -glucosylamidase and e19 -mannosidase. There was lack of enzymatic activity in case of the following hydrolases: e5 lipase (C14), e8 cystine arylamidase, e9 trypsin, e10 -chymotrypsin, e13 -galactosidase, e14 -galactosidase, e15 -glucuronidase, e16 -glucosidase and e20 -fucosidase. On the base of activity of 5 enzymes: e3 esterase (C 4), e7 valine arylamidase, e12 naphthol-as-bl-phosphohydrolase, e16 -glucosidase and e18 N-acethyl- -glucosylamidase, the biotyping of isolated C. albicans strains was made according

5 Charakterystyka cech fenotypowych gatunkowych i wewn¹trzgatunkowych szczepów grzybów wyizolowanych od pacjentów z niewydolnoœci¹ nerek poddanych hemodializom Table V: Hydrolytic activity of C. humicola and C. glabrata strains isolated from faeces Tabela V: Aktywnoœæ enzymatyczna szczepów C. humicola i C. glabrata wyizolowanych z ka³u Enzyme Enzym Mean activity±s [nmoles of hydrolysed substrate] Œrednia aktywnoœæ±s [nmole rozk³adanego substratu] C. humicola C. glabrata [n=6] [n=5] 30.8± ±4.90 e2 Phosphatase alkaline Fosfataza zasadowa e3 Esterase (C 4) / Esteraza (C 4) 7.50± ±3.67 e4 Esterase lipase (C 8) 5.00± ±0.00 Lipaza esterazowa (C 8) e5 Lipase (C 14) / Lipaza (C 14) 0 0 e6 Leucine arylamidase 15.0± ±3.00 Arylamidaza leucynowa e7 Valine arylamidase 3.33± ±1.22 Arylamidaza walinowa e8 Cystine arylamidase 3.33± ±1.22 Arylamidaza cystynowa e9 Trypsin / Trypsyna 0 0 e10 Chymotrypsin/ Chemotrypsyna 0 0 e11 Phosphatase acid 40.0± ±4.00 Fosfataza kwaœna e12 Naphthol-AS-BL-phosphohydrolase 17.5± ±3.67 Fosfohydrolaza naftolowa-as-bl e13 -galactosidase / -galaktozydaza 0 0 e14 -galactosidase / -galaktozydaza 0 0 e15 -glucuronidase / -glukuronidaza 0 0 e16 -glucosidase / -glukozydaza ±1.00 e17 -glucosidase / -glukozydaza 0 0 e18 N-acetyl- -glucosylamidase 2.50± ±1.00 N-acetyl- -glukozylamidaza e19 -mannosidase / -mannozadaza 4.17± ±1.00 e20 -fucosidase/ -fukozydaza 0 0 to Williamson (17) and Kurnatowski et al. (18). It was found that the majority of strains isolated from biological materials from haemodialysed patients belonged to biotype D 3 (acc. to Kurnatowski et al.) 46.8 ± 7.28%, and to biotype E (acc. to Williamson or C 6 acc. to Kurnatowski et al.) 23.4 ± 6.18%. Most frequently C. albicans strains with biotype D 3 were isolated from oral cavity washings 40.4 ± 7.16% (tab. VIII). Other obtained C. albicans strains belonged to biotypes: A, C, F, G, and E (acc. to Williamson); two strains could not be classified to any biotype of previously determined in different investigations (19). Discussion Fungi are often found in biological materials obtained from various ontocenoses. Their presence may be asymptomatic, but they may cause severe, systemic infections in an immunocompromised host. In our investigations the prevalence of fungi in different ontocenoses of patients with renal failure undergoing long-term dialysis was evaluated at the level of 85.7% for the samples collected from oral cavity washings and 50.0% and 7.1% for faeces and urine samples, respectively. There were fungi classified mostly as Candida species: C. albicans, C. glabrata, C. guilliermondii, C. humicola, C. kefyr, C. krusei, C. lambica, C. parapsilosis, C. rugosa, C. tropicalis, C. zeylanoides, and as Cryptococcus albidus, Geotrichum penicillatum, and Saccharomyces cerevisiae. The most frequently isolated fungal species was C. albicans. We found this species in 60.7% and 23.2% of samples from oral cavity and faeces, respectively, but it was absent in urine samples. In studies of intensive care unit patients undergoing urinary catheterization the presence of yeasts was observed in 18.6% of urine specimens (20). C. albicans was recognized in 46.2% of urine specimens; other species: C. glabrata in 30.8% and C. krusei in 7.7%. Species from genus Trichosporon were also found. In patients undergoing permanent immunosuppression after renal transplantation fungal strains were recovered in 65.6% of samples from the oral cavity, 37.5% from rectum and 25.0% from genital organs; C. albicans strains contained 86.4% (15). During the last decade a decrease in prevalence of C. albicans has occurred and an increase in non-albicans Candida species such as C. glabrata, C. krusei and C. parapsilosis is observed (21). In our study C. glabrata was found in 3.57% of samples from oral cavity washings and in 8.93% of samples from faeces. More often C. humicola was found in faeces (10.7%) but it was rarely detected in oral cavity washings (1.7%). In the oral cavity ontocenosis the prevalence of C. tropicalis was the second (5.36%) after C. albicans. Table VI: Percentage of C. humicola strains (n=6) isolated from faeces with enzymatic activity of hydrolases Tabela VI: Odsetek szczepów C. humicola (n=6) wyizolowanych z ka³u wykazuj¹cych aktywnoœæ enzymów hydrolitycznych e ± ± ±19.2 e ± ± e e e ± ± e7 33.3± ± e8 33.3± ± e e ± ± e e e e ± ± e ± ±

6 Characteristics of phenotypic species and intraspecies features of fungal strains isolated from patients with renal failure undergoing... Mikologia Lekarska 2007, 14 (1) Table VII: Percentage of C. glabrata strains (n=5) isolated from faeces with enzymatic activity of hydrolases Tabela VII: Odsetek szczepów C. glabrata (n=5) wyizolowanych z ka³u wykazuj¹cych aktywnoœæ enzymów hydrolitycznych e ± ±21.9 e ± ± e e e ± ± e7 40.0± ± e8 60.0± ± e ± ±17.9 e ± ± e e ± ± e e ± ± e ± ± Table VIII: Biotypes of isolated C. albicans strains (n=47) Tabela VIII: Biotypy wyizolowanych szczepów C. albicans (n=47) Enzyme Enzym Biotypes Biotypy Biotypes acc. to Kurnatowski et. al. Biotypy wg Kurnatowskiego i wsp. Material Materia³ n %±s e3 e7 e12 e16 e18 Oral cavity washings Pop³uczyny jamy ustnej Faeces Ka³ 1. A A ± C B ± F B G C ± E C ± ±4,87 6. D ± ± In our investigation only 8 patients from 56 (14.3%) showed multifocal infections (oral cavity and gastrointestinal tract). In patients after renal transplantation multifocal infections occurred in 68.2% and concerned oral cavity and rectum; in those patients also trifocal infections (genital organs oral cavity rectum) were observed in 12.5% (14). The hydrolytic enzymes activity is considered as one of the most important fungal pathogenicity factor. The majority of strains isolated from oral cavity, faeces and urine in our investigations showed the highest enzymatic activity of the following hydrolases: e11 acid phosphatase, e2 alkaline phosphatase, e6 leucine arylamidase and e12 naphthol-as-bi-phosphohydrolase. The activity of the other enzymes was changing in particular strains. The enzymograms described for strains isolated from the oral cavity, rectum and genital organs of patients after renal transplantation showed also the highest activity in case of e6 leucine arylamidase, e11 acid phosphatase, e12 naphthol-as-bi-phosphohydrolase, and e3 esterase (C4) (12). Interestingly similar results of hydrolytic activity for C.albicans strains isolated from oral ontocenosis of cancer patients were found (22). Results received from the API ZYM test (biomérieux) were used for C. albicans strains biotyping classifying the hydrolytic activity in relation to strains pathogenicity. We found that in biological materials, especially in oral cavity washings, received from patients with renal failure undergoing haemodialysis the most frequently biotype D 3 was detected. Evaluation of enzymatic biotypes of C. albicans strains obtained from healthy persons and patients showed great differentiation of the strains. ukaszuk et al. (22) from the oral cavity of patients with cancer, the most frequently cultured strains from biotype A (67.4%). Krajewska-Ku³ak et al. (19) found C. albicans strains with biotype F (33.7%) and A (30.4%) in mouth washings of patients with candidosis. In other investigations concerning children with suspicion of enteropathy or inflammation of gastrointestinal mucosa, the most frequently found biotypes were: C 6 (30.4%), B 4 (22.3%) and A (16.1%) (18). Analysis of correlation of C. albicans strains biotypes and their resistance to drugs showed the greatest resistance to itraconazol in case of strains isolated from urethra with biotypes I and G, and the greatest resistance to ketoconazol and miconazol in strains with biotypes E and F (19). Such strains were also detected in our investigations: biotype E 23.4 (±6.18)%, and in slight percentages biotypes G (4.25±1.49%) and E (%). Conclusions 1. Prevalence of fungi in patients with renal failure undergoing long-term dialysis was evaluated at the level of 85.7% for the samples collected from oral cavity washings and 50.0% and 7.1% for faeces and urine samples, respectively. 2. The most frequently isolated pathogen was Candida albicans 60.7% and 23.2% from oral cavity and faeces, respectively.

7 Charakterystyka cech fenotypowych gatunkowych i wewn¹trzgatunkowych szczepów grzybów wyizolowanych od pacjentów z niewydolnoœci¹ nerek poddanych hemodializom 3. Strains isolated from oral cavity, faeces and urine showed the highest mean enzymatic activity of the following hydrolases: e11 acid phosphatase, e2 alcaline phosphatase, e6 leucin arylamidase and e12 naphthol-as-bl-phosphohydrolase. 4. The lowest mean enzymatic activity was revealed by hydrolases: e13 -galactosidase, e14 -galactosidase, e16 -glucosidase, e17 -glucosidase and e18 N-acethyl- - -glucosylamidase in case of samples collected from oral cavity and hydrolases: e16 -glucosidase, e17 -glucosidase, e18 N-acethyl- -glucosylamidase and e7 valine arylamidase, e18 N-acethyl- -glucosylamidase, e19 -mannosidase for samples obtained from faeces and urine. 5. The majority of C. albicans strains isolated from different biological materials were classified to biotype D 3 (40.4±7.16%). References 1. Fernandez-Fresnedo G., Ramos M.A., Gonzalez-Pardo M.C., Martin de Francisco A.L., Lopez-Hoyos M., Arias M.: B lymphopenia in uraemia is related to an accelerated in vitro apoptosis and dysregulation of Bcl-2. Nephrol. Dial. Transplant., 2000, 15, Lichodziejewska-Niemierko M.: Zara enia grzybicze. [in]: Dializoterapia w praktyce lekarskiej. ed. Rutkowski B. MAKmedia, Gdañsk 2004, Greenwald B.D., Tunkel A.R., Morgan K.M.: Candida endophthalmitis after lithotripsy of renal calculi. South Med. J., 1992, 85, Westh H., Morgensen P.: Extracorporal shock wave lithotripsy of a kidney stone complicated with Candida albicans septicaemia and endophthalmitis: case report. Scand. J. Urol. Nephrol., 1990, 24, Tapper-James L., Alfred M.J., Walker D.M.: Candidal infections and populations of Candida albicans in mouths of diabetics. J. Clin. Pathol., 1981, 34, Vazquez J.A., Sobel J.D.: Fungal infections in diabetes. Infect. Dis. Clin. Norph. Am., 1995, 9, Abbott K.C., Hypolite I., Tveit D.P., Hshieh P., Cruess D., Agodoa L.Y.: Hospitalizations for fungal infections after initiation of chronic dialysis in the United States. Nephron, 2001, 89, Perfect J.R., Cox G.M.: Fungal infections in diabetic patients and patients with renal failure. Curr. Treat. Opt. in Infect. Dis., 2001, 3, Cheng I.K., Fang G.X., Chan T.M, Chan P.C., Chan M.K.: Fungal peritonitis complicating peritoneal dialysis: report of 27 cases and review of treatment. Q. J. Med., 1989, 71, Kerr C., Perfect J.R., Gallis H.A., Craven P.C., Jorgensen J.H., Drutz D.J., Shelburne J.D.: Fungal peritonitis in patients on chronic continous ambulatory peritoneal dialysis. Ann. Intern. Med., 1983, 99, Nagappan R., Collins J.F., Lee W.T.: Fungal peritonitis in continuous ambulatory peritoneal dialysis: the Auckland experience. Am. J. Kidney Dis., 1992, 20, Bishop M.C.: Infections associated with dialysis and transplantation. Curr. Opin. Urol., 2001, 11, Kurnatowska I., Chrzanowski W., Kacprzyk F., Zamojska S., Kurnatowska A.: Prevalence of multifocal fungal infections in patients undergoing permanent immunosuppression after renal transplantation. Wiad. Parazyt., 2002, 48, Kurnatowska A., Kurnatowska I., Kacprzyk F., Chrzanowski W.: Characteristics of phenotypic specific and intraspecific features of fungal strains isolated from the organ ontocenoses in patients after renal transplantation. Wiad. Parazyt., 2002, 48, Abbott K.C. et al.: Hospitalizations for fungal infections after renal transplantation in the United States. Transpl. Infect. Dis., 2001, 3, Paya C.V.: Fungal infections in solid-organ transplantation. Clin. Infect. Dis., 1993, 16, Williamson M.I., Samaranayake L.P., McFarlane.: Biotypes of oral Candida albicans and Candida tropicalis isolates. J. Med. Vet. Mycol., 1986, 24, Kurnatowski M., W¹sowska-Królikowska K., Toporowska-Kowalska E., Kurnatowska A.: W³aœciwoœci biochemiczne grzybów izolowanych z ontocenoz przewodu pokarmowego dzieci. Wiad. Parazyt., 2001, 47, Krajewska-Ku³ak E., ukaszuk C., Niczyporuk W., Bartoszewicz M., Roszkowska I., Szczurzewski M., Trybu³a J.: Biotypy enzymatyczne szczepów grzybów dro d opodobnych izolowanych z ró nych ontocenoz. Mikol. Lek., 2001, 8, Febre N., Silva V., Medeiros E.A.S.: Microbiological characteristics of yeasts isolated from urinary tracts of intensive care unit patients undergoing urinary catheterization. J. Clin. Microbiol., 1999, 37, Segal E.: Candida, still number one what we know and where are we going from there? Mycoses, 2005, 48, Suppl. 1, S3-S ukaszuk C., Krajewska-Ku³ak E., Niczyporuk W.: Variations of enzymatic activity and biotypes of the yeast like fungi strains isolated from cancer patients. Ann. Acad. Medicae Bialostocensis, 2005, 50, Supported by the Medical University of Lodz, project no Received: Approved: