Sensitivity and lower protease activity of Candida albicans species isolated from Egyptian cancer patients after exposure to cytotoxic and/or radiotherapy treatment

Oropharyngeal candidiasis is a common disease among cancer patients receiving chemo or radiotherapy which precede systemic candidemia, a life threatening infection. This study investigated the diversity and prevalence of different Candida species among Egyptian cancer patients, evaluated the sensitivity of Candida albicans to the frequently administered antifungal therapies and the effect of different radio and chemotherapeutic agents on its virulence. A total of 119 Candida spp. isolates were identified out of 399 clinical samples, of which 72 isolates were C. albicans, 15 were Candida tropicalis, 22 were Candida krusei, and 10 were Candida glabrata. 98.6% of the C. albicans isolates were sensitive to fluconazole; on the other hand, only 8.3% out of the tested isolates were sensitive to amphotericin B. No significant differences were observed in the ability of biofilm formation among C. albicans isolates exposed to chemo, radio or both therapies when compared with standard C. albicans ATCC 60193. Surprisingly, the protease activities in isolates obtained from cancer patients were significantly lower than that of the reference strain after exposure to chemo, radio or both therapies. Thus, it is concluded that radio and chemotherapies may not be in some cases a predisposing factor for the virulence of C. albicans strains.

species are common in cancer patients (Ramirez-Garcia et al., 2014).The penetration of this fungal species into the bloodstream and its dissemination, causing candidemia, is a life threatening infection, being responsible for 30 to 50% of mortality rates among cancer patients (Nucci and Marr, 2005).
Candida albicans is the main resident flora of the digestive mucosa and the genital area, identified in approximately 10 to 20% of healthy adults, followed by Candida glabrata and Candida tropicalis (Zadik et al., 2010).C. albicans is responsible for about 80% of oropharyngeal infections (Bensadoun et al., 2011).Oropharyngeal candidiasis (OPC) is common among patients undergoing intensive chemo or radiotherapy and is known to precede systemic infections (Al-Attas and Amro, 2010).It is a major cause of morbidity in cancer patients (Bodey, 1986).The development of OPC results from the imbalance between fungal virulence factors and host defenses.Several known virulence factors contribute to the pathogenicity of C. albicans, which include adherence to host tissues, phenotypic switching, dimorphism conversion, and enzymes that are integral to its pathogenesis (Calderone et al., 2000;Liu, 2001).Proteinases and phospholipase B are the main enzyme categories secreted by different Candida spp.(De Bernardis et al., 2001).
The common applied therapy, in case of candidiasis, is fluconazole, where its effectiveness is highly proven and superior to other treatments, such as nystatin and clotrimazole with a broad therapeutic range and little toxicity (Sheehan et al., 1999).However, increasing fluconazole resistance in cancer patients is reported (Tortorano et al., 2004).The mechanism of resistance mainly depends on either mutation or over expression of the erg11 gene leading to reduced drug affinity for the target enzyme or increase in ergosterol synthesis, respectively (Lupetti et al., 2002;Maebashi et al., 2003).Caspofungin, is the first member of a new class of antifungal agents targeting the fungal cell wall.It showed high effectiveness in the treatment of candidiasis (Letscher-Bru and Herbrecht, 2003), but unfortunately, its high cost limits its use in Egypt.
Chemotherapy may lead to damage to the mucosal barrier that may result in epithelial atrophy and mucosal ulceration, which may be associated with increased adherence and invasion of Candida (Bensadoun et al., 2011).On the other hand, antineoplastic agents may negatively affect morphogenesis, fungal growth, and virulence of Candida spp. as reported in in vitro studies (Chen et al., 2011).Hence, the present study aimed at the identification of different Candida spp.causing OPC infections and evaluating the sensitivity of C. albicans, the most common infective species in cancer patients, to the frequently administered antifungal therapies and the effect of different chemotherapeutic agents on the virulence of C. albicans.These data are urged to be continuously updated in order to tailor treatment and Khalaf et al. 115 update prevention guidelines.

Sample collection
A total of 399 samples were collected from different cancer patients of both sexes and from adults as well as infants.Patients were submitted in Kasr El-Einy, Center of Radiation, Oncology and Nuclear Medicine, Faculty of Medicine, Cairo University (NEMROCK).All cases were diagnosed by physicians at the hospital.Samples were collected from the patients' oral cavity using sterile sealed swabs.The work was carried in accordance with the code of ethics of the world association (declaration of Helsinki) for experiments involving humans.A written consent was signed by the studied subjects or their parents after full explanation of the study.
The ethical approval was obtained from the medical ethics committee at the Faculty of Pharmacy, Cairo University.The clinical data collected included the personal data, age, residence, clinical diagnosis and predisposing factors, such as diabetes mellitus, pregnancy, use of antibiotics, previous surgical operations, the use of immunosuppressant drugs, exposure to radiation and the type of this radiation.

Identification and maintenance of isolates
All samples were streaked on Sabouraud Dextrose Agar, Oxoid, CM0041 (SDA, pH 6.5) and incubated for 48 h at 37°C.A total of 119 isolates of Candida spp.were identified out of the 399 samples by Gram stain microscopic examination.Further identification was done by isolating the 119 samples on surface of Candida Ident Agar media, modified (Biochemika, Fluka, Sigma Aldrich, 94382), and incubated for 48 h at 37°C.Each isolate represents a unique strain from a single patient.All isolates were scraped from the media, suspended in Brain Heart broth; equal volumes of 30% sterile glycerol were added and mixed evenly using a vortex.The mixtures were then distributed into sterile Eppendorf tubes and stored at -80°C for long time preservation (Prasad et al., 2010).Identified C. albicans samples were subjected to susceptibility, biofilm formation, and protease activity assays in comparison to a standard strain of C. albicans, ATCC 60193.

Susceptibility testing
Antibiotic susceptibility was tested by disc diffusion method described in the Clinical and Laboratory Standards Institute (CLSI).Fluconazole (10 and 25 μg) and amphotericin B (100 units) disks were obtained from Hi-media (India) and Oxoid (Sparks, Md., UK).The standard C. albicans ATCC 60193 and each isolated C. albicans strain were sub-cultured on Sabouraud Dextrose Agar (SDA) plates for 48 h at 37°C.Three to four colonies were transferred aseptically into 5 ml of Sabouraud Dextrose broth and incubated at 25 to 30°C for 8 h.The turbidity of the suspension was adjusted to 0.125 at ʎ = 550 nm which is equivalent to 0.5 Macfarland standard (approximately 1.5 × 10 8 CFU/ml).Plates filled with SDA to a depth of 4.0 mm were used.The SDA surface was inoculated by using a swab dipped in the prepared cell suspension of each isolate in addition to the reference strain, then the antibiotic discs were placed on the surface of the SDA plates.The plates were inverted and incubated at 25 to 30°C for 40 to 48 h.Subsequently, the plates were examined and the zone diameters were measured in mm.The test was repeated twice for each isolated strain and the average diameter was calculated.

Biofilm formation test
C. albicans samples were inoculated in yeast nitrogen base media (YNB) supplemented with 100 mM glucose and cultured overnight.The yeasts were then harvested, washed twice with phosphate buffer saline (PBS, pH 7.2, Ca +2 and Mg free), suspended to 10 7 cells/ml by adjusting the optical density of the suspension to 0.38 at 520 nm, and used immediately.A volume of 100 µl of standardized cell suspension (10 7 cells/ml) of each sample was transferred into sterilized micro titer plate and incubated in a shaker at 75 rpm, for 1.5 h at 37°C to allow the yeast to adhere to the surfaces of the wells.The plate was washed; fresh media was added and incubated again at 37°C in a shaker at 75 rpm.The biofilms were allowed to develop up to 66 h and then the yeasts were quantified by the crystal violet assay as described earlier (Djordjevic et al., 2002).

Quantitative protease assay
Protease activity of C. albicans was assessed by a commercial kit (Pierce Quantitative Protease Assay Kit, Thermoscientific, 23263, Hyclone, USA) according to the method described in Rao et al. (1997) and modified by Tian et al. (2004).The inoculums were adjusted to 0.8 at 550 nm to get 10⁸ cells/ml as described by the manufacturer's protocol.The protease activity was measured using a plate reader at 450 nm.

Statistical analysis
Data of protease activity and biofilm formation experiments were expressed as mean ± standard error of means (SEM).Significant difference was calculated by one way analysis of variance (ANOVA) for both biofilm formation test and quantitative protease assay using Graphpad prism 5 for windows.Differences in results with p<0.05 were considered to be significant.The demographic distribution data were analyzed using Chi square test.The data were expressed as absolute count number.Significance at p value<0.05 was considered.1).Of the C. albicans identified patients, 32 received chemotherapy before sampling and 14 patients were on radiotherapy, while 24 patients received both chemo-and radio-regimens.Two patients did not receive either chemo or radiotherapies and were referred to as unknown treatment in the present study.

Demographic distribution of samples
Figure 1 shows the incidence and prevalence of each Candida spp.among haematological and solid cancer types.It was observed that C. albicans is more prevalent in solid cancer types (64%) followed by C. krusei, then C. glabrata and finally C. tropicalis.Moreover, in the haematological cancer, C. albicans was also the most prevalent (53%), followed by C. tropicalis, C. krusei and C. glabrata.The prevalence of C. albicans and C. krusei was much higher in solid cancer types than in haematological cancer, while the opposite is true for C. tropicalis and C. glabrata.
Figure 2 shows the incidence and prevalence of each Candida spp.among haematological and solid cancer types in relation to different cancer treatments.It was observed that in chemotherapy, the heamatological cancer type was more prevalent than the solid cancer type in all Candida spp.While in radiotherapy, only solid cancer type was observed in all Candida spp.except C. glabrata which was found neither in solid nor in heamatological cancer types.

Susceptibility testing
For fluconazole (10 and 25 µg), 98.6% of the isolated C. albicans were sensitive, 1.4% were of intermediate susceptibility and none of the samples showed resistance.Trailing phenomena was observed and  2.

Biofilm formation
The biofilm formation of C. albicans isolates from each of the 4 groups of cancer patients was compared to that of the standard ATCC 60193.No significant differences were observed among chemotherapy (0.449±0.002), radiotherapy (0.450±0.003), both therapies (0.446±0.001), or unknown treatment (0.459±0.01) groups as compared to the standard ATCC 60193.Results are demonstrated as show in Figure 3.

Protease production
The protease concentrations in C. albicans isolates obtained from cancer patients were significantly lower  than that of C. albicans ATCC 60193 reference strain after exposure to chemotherapy, radiotherapy, both treatments, or unknown treatment (Table 3).

DISCUSSION
Patients who undergo chemo and/or radiotherapy are at increased risk of developing fungal infection.In the case of changes in the mucous membrane, fungi can move into the blood and develop into disseminated fungal infection, often leading to death.Hence, it is important to determine  Tudela et al., 2002).Furthermore, most of the OPC samples from cancer patients in different regions were also identified as C. albicans with a range of 33 to 76% of the total isolates (Laverdiere et al., 2002;Al-Abeid et al., 2004;Belazi et al., 2004).This study also showed higher prevalence of C. albicans in both solid and haematological cancer types, followed by C. krusei, then C. glabrata and finally, C. tropicalis in solid cancer types.While in the haematological cancer types, C. albicans was found to be followed by C. tropicalis, C. krusei and C. glabrata, respectively.The prevalence of C. albicans and C. krusei was much higher in solid cancer types than in haematological cancer, while the opposite is true for C. tropicalis and C. glabrata.This was also the case in a study made by Slavin et al. (2010) where the prevalence of C. ablicans in solid organ malignancies was higher than in haematological malignancies, 51 and 33%, respectively.
The present study showed that chemotherapy discloses the most incidence of C. albicans infection (44%), followed by the combined chemo-radiotherapy, a result that coincides with the study made by Al-Abeid et al. (2004) where the chemotherapy patients were colonized more frequently by C. albicans than radiotherapy patients.
The adherence level of the studied isolates was found not to be significantly different from that of ATCC control strain.This result does not agree with that obtained from the study made by Al-Abeid et al. (2004), where there was a statistically significant difference between the number of adhered C. albicans and the ATCC control strain.
Our findings also revealed that the protease production was lowered in isolates from different cancer patients than that of ATCC control strain.The same result was suggested by Al-Abeid et al. (2004).This may be explained by the fact that, some cytotoxic agents can lower the protease production in C. albicans as various anticancer agents, such as glucocorticoids, cytotoxic agents and calcineurin inhibitors have direct inhibitory effects on and/or have altered the biology of fungal cells and in some cases can also be used as a combined therapy with antifungal agents (Chen et al., 2011).Although the virulence of the C. albicans is lowered, the infection and colonization persists in cancer patients, this may be due to the immune-suppression effect of anticancer treatments.
Disk diffusion method was used to determine the susceptibility of C. albicans to fluconazole and amphotericin B as it represents a reliable and reproducible method (Rodri´Guez-Tudela et al., 1996).C. albicans isolates in the current investigation were susceptible to fluconazole and of intermediate sensitivity to amphotericin B suggesting the prime role the fluconazole has in fungicidal activity.These results are in accordance with other studies reporting susceptibility of C. albicans in cancer patients (Al-Abeid et al., 2004;Slavin et al., 2010).Additionally, it was noted that strains exhibiting trailing growth, as seen in the present experiment, responds to low doses of fluconazole (Revankar et al., 1998).
In conclusion, the effective management of C. albicans infections prior to any anti-cancer treatment is highly recommended in order to avoid the complications that might arise from the fungal infection upon starting the anticancer treatment.Although the current study indicates that cancer therapy, either chemotherapy or radiotherapy, has affected the virulence of C. albicans, it is highly recommended to keep caution when dealing with powerful antifungal agents in the prophylaxis or the management of OPC especially in immune-compromised patients to avoid the possible emergence of resistant strains.

Figure 1 .Figure 2 .
Figure 1.Distribution of Candida spp.among different cancer types.Significance is calculated using Chi square test P value<0.05.

Figure 3 .
Figure 3. Biofilm formation of C. albicans among different therapy groups.Data represent mean ± standard error of mean (SEM).Statistical analysis was carried out by one-way analysis of variance (ANOVA).

Table 1 .
Distribution of Candida spp.according to age and sex of patients.

Table 2 .
Susceptibility testing of isolated C. albicans strains by disc diffusion method.

Table 3 .
Protease enzyme levels of C. albicans in cancer patients received chemotherapy, radiotherapy or both, compared to C. albicans ATCC 60193 reference strain.presence of fungi in this group of patients before the beginning of chemo-or radiotherapy to enable early treatment.The current study discussed the prevalence and the virulence of C. albicans among this group of patients.A total of 60.5% of candidiasis subjected in this study was identified as C. albicans, which is a large proportion despite the widespread use of fluconazole.On the other hand, other Candida spp.were found in minor proportions.This complies with a previous report, where C. albicans constituted 56.3% of patients receiving radiotherapy for head and neck neoplasms, while each of C. glabrata and C. tropicalis was present in 12.5% of the total patients ( the