Emilija Mlinarić-Missoni

Smilja Kalenić

Verica Verica Važić-Babić

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219Acta Dermatovenerol Croat          2006;14(4):219-224        ORIGINAL SCIENTIFIC ARTICLEBiotypes of Candida albicans Isolated from Cardiovascular System and Skin Surveillance Cultures of Hospitalized PatientsVerica Važić-Babić1, Emilija Mlinarić-Missoni1, Smilja Kalenić2 1Mycology Department, Croatian National Institute of Public Health, Zagreb, Croatia2Department of  Clinical and Molecular Microbiology, Zagreb University Hospital Center, Zagreb, Croatia   Corresponding author:Verica Važić-Babić, PhDCroatian National Institute of  Public HealthRockefellerova 2HR-10000 Zagreb,  Croatiaverica.vazic.babic@hzjz.hrReceived: July 14, 2006Accepted: September 13, 2006SUMMARY The aim of the study was to biotype 59 isolates of Candida (C.) albicans from cardiovascular system samples (blood and intravenous catheter) and 123 isolates of the same species from skin surveillance cultures (swabs of the armpit, groins and intravenous catheter insertion sites) of hospitalized patients using the Odds and Abbott biotyping method. Biotyping of 59 isolates of C. albicans taken from the cardiovascular system samples revealed the presence of 16 biotypes. Biotype 355 was the most common biotype, accounting for 35.6% of all biotype isolates from this system. Biotyping of 123 C. albicans isolates from skin surveillance cultures detected 21 biotypes. Biotype 355 was most common, accounting for 17.9% of all biotype isolates from these samples. The two systems had 10 biotypes in common: 355, 155, 257, 305, 105, 315, 300, 015, 157, and 345. These biotypes accounted for 88.3% and 81.4% of all C. albicans biotypes isolated from the cardiovascular system and skin surveillance cultures, respectively. Biotypes 355, 155, and 257 were the biotypes most frequently shared in isolates from the two systems. These biotypes accounted for 57.7% and 43.1% of all C. albicans biotypes isolated from the cardiovascular system and skin surveillance cultures, respectively. KEY WORDS: biotype; Candida albicans; cardiovascular system; skin surveillance cultureINTRODUCTION Candida (C.) albicans is ubiquitous commensal yeast in humans in whom it is part of the physio-logical ﬂora of the skin and mucous membranes. In individuals with preserved immunity, it may cause disease exceptionally rarely. When body defenses fail for any reason, this yeast behaves opportu-nistically. As the number of immunocompromised patients across the world is steadily increasing, the incidence of fungal infections follows this trend (1,2). Candida spp. are the most common agents (78.3%) of fungal nosocomial infections, and C. al-bicans has been demonstrated to be the causative agent in more than 50% of these infections (3-5). The ever greater use of intravenous catheters is the reason for this increase in the incidence of fungemia coupled with the use of catheter in im-munocompromised patients. This is due to the 220 ACTA DERMATOVENEROLOGICA CROATICAopportunistic species of the fungus from the pa-tient’s skin and his surrounding (6,7). In 50% to 80% of candidemia in patients with hematologic malignancies, intravenous catheter is the site of Candida introduction into the bloodstream (8). In these patients candidemia may result in the development of severe and often fatal invasive and disseminated candidiasis (endocarditis, en-dophthalmitis, microabscesses in the brain and kidney, etc.) (5,9). In some 50% of immunocom-promised patients, C. albicans is isolated from the skin around the catheter insertion site. In addition to the source of yeast from the patient’s own skin, yeasts from the patient’s environment (e.g., con-taminated objects and infusion solutions as well as the hands of hospital staff and secretions from their respiratory systems) may also lead to coloni-zation of intravenous catheter. This may also oc-cur secondarily by hematogenous dissemination of the yeast from a remote focus of inﬂammation with the same yeast (6,8,10,11). A variety of phenotyping (biotyping, serotyp-ing, morphotyping, enzyme typing, assessment of sensitivity to antimycotics and “killer” toxins, etc.) and genotyping methods have been employed to identify the types of C. albicans (12,13). Results reported by other workers have pointed to the bio-typing method as being highly reproducible and able to differentiate particular types within the C. albicans species (13).  The aim of the present study was to identify the types of C. albicans isolates taken from samples of the cardiovascular system and skin surveillance cultures of inpatients using the Odds and Abbott biotyping method (14,15). This typing method al-lows for identiﬁcation of 512 different biotypes of this species, which enables monitoring of the fre-quency of individual biotypes in different systems of the patient as well as detection of the poten-tial sources of infection. The method is suitable to examine a large number of isolates and does not entail major material expenses or expensive equipment. PATIENTS AND METHODSSamples Fifty-nine isolates of C. albicans from the car-diovascular system (blood and intravenous cath-eter) and 123 C. albicans isolates from skin sur-veillance cultures (swabs of armpit, groins and intravenous catheter insertion sites) were typed. The samples derived from patients hospitalized at Sterile Unit, Department of Hematology, Univer-sity Department of Medicine, Zagreb University Hospital Center. Isolation and identiﬁcation methods All clinical material samples were inoculated onto Sabouraud’s glucose agar and incubated aerobically at 37 ºC for 3 to 7 days. Identiﬁcation of C. albicans was made by standard tests (test of germination, assimilation and sugar fermenta-tion tests, and development of chlamydospores on cornmeal agar) and commercial tests (ID 32 C and API 20 C AUX, BioMerieux) (3,16). Biotyping method The isolates were stored at -26 ºC in glycerin broth until analysis. Resuscitated isolates were typed by using the Odds and Abbott method (14,15). Each isolate underwent testing on nine different media (those to examine its tolerance of pH 1.4 and increased NaCl concentrations, thus testing its ability to excrete proteinase and assimi-late urea, citrates and sorbose, and its resistance to 5-ﬂuorocytosine, boric acid and safranin). The isolate growth on a given medium was considered as positive test outcome; the same person read all test media (Figs. 1-4). Test media were divided into three groups, the biotype of each isolate being marked with a 3-digit number accordingly. Each digit was calculated based on the sum of test re-sults within each group.  RESULTS Table 1 illustrates the frequency of C. albicans biotypes isolated from cardiovascular system. Sixteen biotypes were typed in 59 isolates. The a) b)Figure 1. (a) Multiple inoculator for preparing sus-pension of Candida albicans isolates; (b) multiple inoculator for inoculating Candida albicans iso-lates onto different media.Važić-Babić et al.     Acta Dermatovenerol CroatBiotypes of Candida albicans      2006;14(4):219-224   221ACTA DERMATOVENEROLOGICA CROATICAfour most common biotypes (355, 155, 257 and 305) accounted for 66.2% and the remaining 12 biotypes for 33.8% of all biotypes from the cardio-vascular system samples. The prevalence of bio-type 355 was highest with 35.6% of all biotypes. Biotype 155 had a prevalence of 13.6%, and bio-types 257 and 305 of 8.5% each (Fig. 5).  The frequency of C. albicans biotypes isolated from skin surveillance cultures (armpit, groin and catheter insertion site swabs) is shown in Table 2. Typing of 123 isolates yielded 21 biotypes. The four most common biotypes (355, 155, 257 and 300) accounted for 53.7% of all biotypes from skin surveillance cultures. Biotype 355 was most prev-alent (17.9%), followed by biotypes 155 (13.0%), 257 (12.2%) and 300 (10.6%) (Fig. 6).  Table 3 presents biotypes of C. albicans isolates taken from samples of the cardiovascular system and skin surveillance cultures of 16 hematologi-cal patients. Isolates taken from these samples of each individual patient belonged to one to three biotypes. In 14 (87.5%) patients, the biotypes found in the isolates from cardiovascular system and skin surveillance cultures were the same.Table 1. Frequency of Candida albicans biotypes isolated from cardiovascular system Biotype No. of isolates (%)355 21 (35.6)155 8 (13.6)257, 305 10 (17.0)105 4 (6.8)315, 300 6 (10.2)017 2 (3.4)015, 045, 100, 115, 157, 314, 345, 360 8 (13.6)Total  16 59 (100)Figure 3. Growth of 40 Candida albicans isolates on glycine medium to demonstrate its ability to use glycine as the source of nitrogen and carbon. Table 2.  Frequency of Candida albicans biotypes isolated from skin surveillance cultures Biotype No. of isolates (%)355 22 (17.9)155 16 (13.0)257 15 (12.2)300 13 (10.6)105 9 (7.3)305, 345 16 (13.0)144, 157, 241, 315, 357 20 (16.5)041 3 (2.4)147 2 (1.5)005, 015, 055, 175, 205, 325, 755 7 (5.6)Total 21 123 (100)DISCUSSION  C. albicans isolates typed in the present study originated from samples of the cardiovascular system (blood and intravenous catheter) and of the skin surveillance cultures (armpit, groin, and/or catheter insertion site swabs) from hospital-ized hematological patients. Intravenous catheter was the site of Candida spp. introduction in the bloodstream in 50% to 80% of candidemia, and C. albicans was responsible for 55% of these infec-tions (2,5,6,8,10). In about 50% of immunocom-promised patients, C. albicans was isolated from the skin around the intravenous catheter insertion site (6,7,10,11).  These data have prompted us to attempt typ-ing and comparing the detected types of C. albi-cans isolated from the cardiovascular system and surveillance skin cultures of hospitalized hemato-Figure 2. Growth of 49 Candida albicans isolates on control medium.Važić-Babić et al.     Acta Dermatovenerol CroatBiotypes of Candida albicans      2006;14(4):219-224 222 ACTA DERMATOVENEROLOGICA CROATICAlogical patients. The Odds and Abbott biotyping method was chosen for typing for its simple per-formance and allowing for examination of a large number of samples without entailing great mate-rial expenses or expensive equipment. Although the methods of genotyping are currently also available for microorganism (and yeast) typing, phenotyping methods are still in common use in epidemiological studies of nosocomial infections (17).  Literature reports of research ﬁndings on the biotyping of C. albicans isolates with the Odds and Abbott method are very scarce. Most research-ers typed isolates of this species originating from two systems, genitourinary and digestive systems (14,18). Although this method permits typing of 512 different C. albicans biotypes, the reported typing results have revealed much fewer (from 25 to 79) biotypes (14,18). On typing C. albicans isolates taken from skin surveillance cultures of hematological inpatients, the present study found a greater number of bio-types (n=21) in these than in the cardiovascular system samples from the same patients (n=16). In the isolates from these samples, 10 biotypes were the same: their frequency in the isolates from sam-ples of the cardiovascular system and skin surveil-lance cultures was 88.3% and 81.4%, respective-ly. The present study demonstrated a high coinci-dence (87.5%) between the biotypes recorded in isolates from the cardiovascular system samples Table 3.  Biotypes of Candida albicans isolated from cardiovascular system samples (blood and/or intra-venous catheter) and skin surveillance cultures (swabs of armpit, groin and/or site of catheterization) in 16 hospitalized hematological patientsPatientCardiovascular system Skin surveillance culturesBlood Intravenous catheter Catheterization site   Armpit/groin1 355 3552 155 155 1553 315 300 3054 355 355 3555 357 355 3576 357 3577 155 1558 157 257 157,2579 155 157 155, 15710 355 315 31511 257 25712 300 30013 345 345 34514 305 31515 105 105 10516 157 157Figure 4. Growth of 43 Candida albicans isolates on safranin medium to demonstrate resistance to safranin stain.35,60%13,60%8,50%8,50%33,80%biotype 355biotype 155biotype 257biotype 305other 12 biotypesFigure 5. Prevalence of the four most common Candida albicans biotypes isolated from the car-diovascular system. Važić-Babić et al.     Acta Dermatovenerol CroatBiotypes of Candida albicans      2006;14(4):219-224 223and skin surveillance cultures in 16 hematological inpatients. Klempp-Selb et al. obtained compara-ble results. They typed chromosomal DNA of 19 C. albicans isolates taken from blood samples and 36 C. albicans isolates taken from other samples (stool, urine, sputum, vaginal swabs, and swabs of the skin around the site of central venous catheter insertion) from six hospitalized patients.  Karyo-typing revealed the presence of 10 different karyo-types. In 5 (83.3%) patients, the karyotypes in the isolates from blood and other samples proved to be the same (19). Using the “killer-yeasts” typing method, Candido et al. typed 146 C. albicans iso-lates taken from various samples of hospitalized patients, i.e. blood, stool, bronchoalveolar lavage, urine, swabs of the oral cavity, vagina, skin lesions and intact skin). Typing detected 23 biotypes, the most common being 211, 111 and 811. Type 211 was most common among the isolates from blood samples (20). Betremieux et al. typed C. albicans isolates obtained from blood samples of seven premature infants admitted to intensive care unit (ICU), and C. albicans isolates from swabs of the skin of hospital staff hands. Five methods (se-rotyping, morphotyping, resistance typing, “kill-er-yeast” typing, and genotyping) were used on typing. All blood sample isolates were of the same type as the isolate from the skin of the hand of an ICU worker, indicating the role of yeasts from the patient’s surrounding in intrahospital candidemia (21).  CONCLUSION Biotyping of 59 C. albicans isolates obtained from the cardiovascular system of hospitalized he-matological patients showed 16 biotypes. Biotyp-ing of 123 C. albicans isolates from surveillance cultures of the skin of the same patients revealed 21 biotypes. Three most common biotypes (355, 155 and 257) accounted for 57.7% of all C. albi-cans biotypes isolated from the cardiovascular system and 43.1% of all biotypes of the C. albicans isolates from surveillance cultures of the skin. The results of our study showed C. albicans to be not only a major colonizer of the skin and intravenous catheters in hematological inpatients, but also an agent of candidemia in this population. Along with other phenotyping and genotyping methods, the Odds and Abbott biotyping helps in differentiat-ing particular types within the C. albicans species, permitting detection of the epidemiologic sources of nosocomial infections and routes of spread of this yeast.  References1.   Raad II. 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Pediatr Infect Dis J 1994;13:899-905. Važić-Babić et al.     Acta Dermatovenerol CroatBiotypes of Candida albicans      2006;14(4):219-224 Elida cream, for the use every hour; year 1935.(from the collection of Mr. Zlatko Puntijar)

Biotypes of Candida albicans Isolated from Cardiovascular System and Skin Surveillance Cultures of Hospitalized Patients

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