Candidiasis, caused by Candida spp, is the most common fungal infection in immunocompromised patients, resulting from overuse of antibiotics, immunosuppressive drugs in chemotherapy, organ transplantation, HIV/AIDS, and diabetes. All of these factors contribute to an increased incidence of candidiasis [1]. The global incidence of candidiasis has reached 750,000 cases annually [2], with approximately 200 species of yeast documented, including 20 pathogenic species for humans. However, 90% of candidiasis infections occur due to common species such as C. albicans, C. krusei, C. tropicalis, C. glabrata, and C. parapsilosis. These yeasts are part of the normal flora, living naturally in various sites of the human body. However, when the body's immune system is compromised, they transform into pathogenic fungi [3]. These species are characterized by possessing various virulence factors that facilitate their spread and infection of different sites in the human body [4].

Chronic kidney disease (CKD) is a condition in which the kidneys lose their ability to function normally. It can be caused by several factors, including infections, autoimmune diseases, diabetes, other endocrine disorders, cancer, and exposure to harmful chemicals [5]. Patients with chronic kidney failure undergoing regular dialysis sessions are more susceptible to opportunistic fungal infections, which can lead to bloodstream infections and are considered one of the leading causes of death in acute kidney failure patients [6,7]. They also suffer from a high incidence of anemia, malnutrition, and multifactorial immune deterioration [8]. On the other hand, hemodialysis (HD) is associated with long-term immune system suppression, affecting both cellular and humoral immunity [9,10]. Uremia is also linked to reduced phagocytic activity, increased programmed cell death in neutrophils, and depletion of regulatory T cells [11], making the immune system less effective and more susceptible to fungal infections [8].

The use of antifungal agents in humans can have side effects, including fever, chills, high blood pressure, and potential toxicity [12]. The role of antifungal agents can be twofold, either killing fungi or inhibiting their growth, but this effect may be limited to specific groups of fungi and not all types. Most antifungal agents currently used affect either the biosynthesis of the fungal cell wall or interfere with pyrimidine metabolism or interfere with sterol metabolism [13]. The immune mechanisms for defense against fungal infections are diverse, ranging from ancient protective mechanisms   that   are  specifically    triggered    during   

infection and disease [14]. So this result aimed to evaluate the sensitivity of the Candida spp to the some antibiotic. 

Sampling

One hundred and ninety urine samples were collected from chronic dialysis patients in addition to 20 urine samples as control groups of both gender and different ages at Kirkuk Teaching Hospital in Kirkuk city, during the period from November 2022 to March 2023.

Isolation of Candida spp

        All urine samples were cultured on SDA media, and incubated at 37 ℃ for 48 hours. Identified all fungal growth depended, color, size and morphology of the colonies [15].

Identification methods

Morphological Identification Candida spp.

Candida spp isolates were identified based on the developing colonies on SDA and that appear as smooth, glabrous, yeast-like structures that are white to cream in color [16].

Microscopically identification Candida spp

A portion of the fungal colony was taken and mixed with a drop of lactophenol cotton blue stain, then spread onto a glass slide. The sample was then examined under a light microscope at magnifications of X10 and X40 to observe pseudohyphae and budding [17].

CHROMagar Candida Media

Chrome Candida agar is a selective medium (Himedia, india) that is utilized for the identification of Candida spp. Samples were cultured on this medium, and each species displays a distinctive color. Petri dishes containing the cultured samples are then incubator at 37 ℃ for 48 hours. In order to identify the different the species of Candida [18].

Germ Tube Test

The method involves taking a portion of the colony and placing it in a test tube containing 0.5 ml of blood serum. The mixture is then incubated at 37 ℃ for 3 hours. After that, a drop of the suspension is taken and placed on a glass slide and examined under a microscope to observe the germ tube. This examination is distinctive for the C. albicans, C. dubiniensis, and C. stellatoidea [19].

Antifungal susceptibility test

The sensitivity test for yeast was performed using the disk diffusion method according to the Clinical and Laboratory Standards Institute (CLSI) guidelines, different antifungal agents and concentrations was used, including Nystatin (NY), Ketoconazole (KCL), Clotrimazole (CLO), Fluconazole (FLU), and Amphotericin B (AMB) (Liofilchem, Italy). A yeast suspension was prepared for each isolate, and the turbidity was measured to be between 1.8-2.20 using a McFarland. The suspension was then streaked onto the surface of SDA medium using a sterile loop. Antifungal disks were placed on the surface of the medium and then incubated at 37°C for 24-48 hours, with growth monitored. The results were recorded, and the diameters of the inhibition zones around the different antifungal disks were measured using a ruler [20].

The current study showed that females are more susceptible to Candidiasis compared to males and the percentage was 58.7%, 41.3% respectively. The highest percentage of Candidiasis occurred in patients age groups between 41-50 years (Table 1), which is in line with [21,22,23] who reported that most affected patients were over 45 years old. However, there is still no definitive relationship due to differences in population and racial samples. Renal insufficiency is associated with decreased immune function, leading to increased susceptibility to infections in these patients and immune activation, leading to inflammation [24, 25]. The results of urine culture on SDA media showed 46 positive samples, accounting for 24.2%, and 144 negative samples, accounting for 75.8%. No fungal growth was observed in the control samples (Figure 1).

The results of laboratory culture on differential medium CHROMagar Candida that used to identify Candida spp based on colony color indicated five species of the Candida. C. albicans, C. glabrata, C. kruzei, C. parapsilosis and C. trpicals as showen C. albicans was the most common isolate, accounting for 41.3% of isolates, characterized by green colonies on CHROMagar Candida. All C. albicans exhibited germ tube formation. This was followed C. glabrata 30.4 % with dark purple color, C. krusei 17.4 % with light pink color, C. parapsilosis 6.5 % with white color, and C. tropicalis 4.4 % with blue colonies. Non-albicans species were unable to form germ tube formation. These findings are consistent with the results of [26, 22], and [27, 28], but do not agree with the results of [29]. This phenomenon can be attributed to several reasons, including immunocompromised of the patients, making them more susceptible to urinary tract infections [30]. Additionally, the absence of a natural voiding mechanism plays a mechanical role in expelling pathogenic organisms. Furthermore, impaired kidney function leads to the inability to excrete substances in the urine that typically act as antimicrobial factors [31] (Table 2, Figure 2, 3).

Table 1: Demographic description of the study sample.

Figure 1: Percentage of Candida spp. isolated from urine samples

Table 2: The morphological and biochemical characteristics, of the isolated candida species.

*-: Negative, +: Positive

A study of antifungals activity conducted on five species of the most frequent Candida spp recognized as causative agents of Candidiasis C. albicans, C. glabrata, C. krusei, C. parapsilosis, C. tropicalis, by using the disk diffusion method and the study results showed that Ketoconazole was the most effective antifungal agent against Candida species, followed by Clotrimazole and Fluconazole (see Figure 4 and Table 3, 4). These results are consistent with [32] and [33]. However, although Azole antifungal agents are effective in inhibiting yeast growth, they Amphotericin B, which is consistent with [34] and [32]. They reported that Candida species C. glabrata, C. krusei, and C. parapsilosis were 100% resistant to Nystatin. However, these findings do not agree with [35], who reported that Candida species C. glabrata, C. krusei, C. parapsilosis, C. tropicalis, and C. albicans were sensitive to Nystatin. The results also did not agree with those of [36] when studying have negative side effects on the liver, as reported by [37]. On the other hand, the Candida species showed resistance to Nystatin and Candida spp, as they found that they were  sensitive    to   Amphotericin  B.

The azoles target is to act on inhibition of the enzyme cytochrome P450 oxidase, preventing the formation of an important compound called ergosterol in the fungal cell wall. This can cause gaps or tears in the cell   wall.    Additionally,   this inhibition   makes  the Oxidation process to be disabled in the plasma membrane. The accumulation of phospholipids within the cell due to the effect of the antifungal agent on membrane composition can also contribute to fungal cell death [38].

Sensitivity test results for yeast can vary due to several factors, including the studied population, previous treatments taken by the patient, the patient's internal characteristics, excessive or incorrect use of antibiotics, and an increase in immunocompromised patients [39]. Other factors that may affect the results of antifungal sensitivity tests include the inoculum size, incubation period and temperature, and the medium used in the test [40]. The sensitivity test for antifungal agents plays a vital role in selecting the appropriate antifungal agent for treating fungal infections [41], which reduces morbidity and mortality rates among patients. Therefore, antifungal agents should be carefully selected after conducting accurate analysis and confirming the diagnosis [42]. The indiscriminate and frequent use of antibiotics is considered to contribute to the emergence of new resistant strains of Candida spp. For this reason, it is expected that these species of Candida spp. may differ from one type to another. Additionally, these species may also vary based on the source from which they were collected [43].

Figure 2: Candida spp on Chrom Candida Agar and SDA, A C. albicans, B C. glabrata, C C. tropicalis, D C krusei and E C. parapsilosis

Figure 3: C. albicans germ tube formation 40x

Figure 4: The effect of antifungal on candida spp.

Table 3: Antifungal used of Studied samples

Table 4: The sensitivity and resistance of isolates of fungal species

KCA: Ketoconazole, FLU: Fluconazole, CLO: Clotrimazole, CC: Clotrimazole, AMB: Amphotericin B, NY: Nystatin, S: sensitivity, R: Resistance

The study focused on the causative agent of candidiasis in patients with chronic dialysis patients in the city of Kirkuk- Iraq, and isolated five of Candida species. The Candida albicans was the most common species compared to other isolated. And the use of medical device used for hemodialysis increase the risk of fungal infection.

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