Species distribution and antifungal susceptibility patterns of oral Candida colonization and infection in β-thalassemia major patients

Abstract

Background

Patients with β-thalassemia major often have immune dysfunctions that heighten their risk of oral Candida colonization and infection. This study aimed to assess the prevalence of oral Candida, including Candida albicans and non-Candida albicans, and evaluate their antifungal susceptibility profiles.

Methods

A total of 270 patients were enrolled, and oral swabs were collected from the gums, teeth, tongue, palate, and cheek mucosa. Species identification was performed by combined phenotypic characteristics (CHROM agar Candida) and molecular assay (PCR-RFLP and sequencing), and antifungal susceptibility testing was performed by CLSI M38 broth microdilution.

Results

A study of 270 patients revealed that 17 (6.29%) had oral candidiasis, and 22 (8.14%) showed Candida colonization. The most common species were Candida albicans (52.38%), Pichia kudriavzevii (38.09%), and Nakaseomyces glabratus (9.52%). In colonization cases, the predominant species were C. albicans (92%) and P. kudriavzevii (8%). Notably, multidrug-resistant C. auris was absent. Voriconazole and ketoconazole were the most effective antifungals, while nystatin and fluconazole had lower efficacy.

Conclusion

The increasing use of prophylactic antifungal agents, together with emerging resistance among Candida species, underscores the importance of early detection and effective management of fungal infections in high-risk populations such as patients with ß-thalassemia. Regular oral examinations and the use of reliable diagnostic methods are essential for timely identification and treatment. Although multidrug-resistant Candida isolates have not yet been reported in this group, continued surveillance remains crucial for infection control within healthcare settings. Further research is needed to clarify the epidemiology of fungal colonization and infection in ß-thalassemia patients and to determine the clinical significance of isolating these organisms.

Introduction

Thalassemia is a genetic blood disorder resulting from genetic mutations that affect either the alpha- or beta-globin protein chains, leading to hemolytic anemia classified into two types: major (homozygous) and minor (heterozygous). Among these, β-thalassemia major represents the most severe form of inherited hemolytic anemia [1, 2]. However, thalassemia has recently been classified into non-transfusion-dependent thalassemia (NTDT) and transfusion-dependent thalassemia (TDT), based on the necessity for regular blood transfusions. Most patients with severe β-thalassemia major require lifelong regular blood transfusions to survive, and their treatment typically includes iron chelation therapy and splenectomy [2, 3]. Immunologic defects have been identified in patients with thalassemia, particularly after splenectomy. In non-splenectomized thalassemia patients, levels of all immunoglobulin classes (IgG, IgA, and IgM) are elevated; however, IgM levels decrease following splenectomy [4]. The World Health Organization (WHO) has recognized thalassemia as a significant public health concern and estimated that 7% of the global population is a carrier, with 80% residing in developing countries [5]. Iran is one of the major centers for the prevalence of β-thalassemia major in the eastern Mediterranean region. Estimates suggest between two and three million β-thalassemia major carriers and 25,000 patients are in Iran [6]. Infectious diseases are a common complication in patients with thalassemia major and are the second leading cause of death, following heart failure, accounting for 12–26% of patient deaths [3, 7]. The most commonly reported infections in patients with beta-thalassemia major are often transfusion-transmitted viral infections and severe bacterial infections [3, 8–11]. However, patients with thalassemia major may also be vulnerable to fungal infections. These infections can be hazardous due to factors like iron overload, immune dysfunction, and the effects of splenectomy, which are common in thalassemia management [12–15]. Candida species are opportunistic microorganisms commonly present in the oral, gastrointestinal tracts, and skin as part of the normal flora. However, alterations in the host’s microenvironment and compromised immune defenses can trigger the transition of Candida species from harmless commensals to pathogenic organisms, leading to a wide range of infections, ranging from superficial mucocutaneous to life-threatening disseminated candidiasis [16, 17]. While Candida albicans has long been regarded as the primary species responsible for candidiasis in humans, there has been a notable increase in infections caused by non-albicans Candida species over the past two decades [18]. The increase in colonization and infections by non-albicans Candida species could be related to factors consisting prior exposure to polyene and azole drugs, widespread use of broad-spectrum antibiotics and antifungal agents, a rise in individuals with immune system deficiencies, long term use of catheters and medical devices, prolonged treatment with immunosuppressive and cytotoxic medications, aging populations, hematopoietic stem cells and solid organ transplantations, advancements in molecular diagnostic methods, and other associated factors [19–21]. Remarkably, in recent years, Candida auris, a multidrug-resistant fungal pathogen primarily associated with healthcare settings, has emerged as a significant global health threat due to its broad resistance to antifungal compounds, its persistence on human skin and in the environment, and its capacity to spread within long-term care facilities and hospitals [22]. Prolonged hospitalization, blood transfusions, and the use of indwelling catheters can serve as risk factors for C. auris infections in patients with underlying conditions such as β-thalassemia [23, 24]. Therefore, screening patients for non-albicans Candida colonization enables healthcare facilities to implement appropriate infection prevention and control measures, thereby reducing the risk of transmission and infection [25, 26]. Although approximately 60% of healthy individuals harbor Candida species as part of their normal oral flora (harmless commensal organisms), Candida can potentially cause infections, especially in immunocompromised individuals, when it overgrows. Oropharyngeal candidiasis is an opportunistic mucosal fungal infection of the oral cavity resulting from an overgrowth of Candida yeast. It is often associated with conditions such as immunosuppression, diabetes, use of broad-spectrum antibiotics, and corticosteroid therapy. Numerous studies have reported a higher prevalence of oral candidiasis in cancer patients, diabetic patients, and individuals with HIV infection compared to healthy individuals [27–30]. Patients with thalassemia major possess a multifactorial predisposition to Candida infections. Immune dysregulation, encompassing neutrophil dysfunction, T-lymphocyte depletion, and post-splenectomy alterations, combined with iron overload, frequent and prolonged hospitalizations, broad-spectrum antibiotic exposure, and the use of central venous catheters, collectively establishes a permissive microenvironment that is conducive to fungal colonization and invasion [31–34]. With this background in mind, the present study aimed to evaluate the prevalence of oral Candida colonization and infections among patients with β-thalassemia major registered at the Thalassemia Center in Mazandaran Province. Furthermore, it sought to screen for C. auris, analyze species distribution, and assess the antifungal susceptibility profile of the isolates.

Methods

Study participants

Patients with thalassemia major who attended the Thalassemia Research Center at Bu Ali Sina Hospital in Sari, Iran, participated in this study conducted between April 2024 and April 2025. The study’s objectives were clearly explained to the participants, and all individuals provided written informed consent. To be included in the study, participants had to meet specific criteria: they must be patients who frequently receive blood transfusions (transfusion-dependent thalassemia) and be at least 8 years old. Exclusion criteria included individuals unwilling to cooperate, those who had taken antifungal medications, patients who used a toothbrush prior to sampling, and individuals with dentures. Data collected included gender, age, history of splenectomy, use of oral antibiotics or antifungal medications, diabetes status, and the type of chelating therapy employed.

Sample size calculation

Based on the previous studies in similar immunocompromised cohorts and regional data [31, 34], the prevalence of oral Candida colonization/infection was estimated 30% (p = 0.30). Then, using the following formula and considering a type I error of 0.05 () and an error limit of 0.06 (d = 0.06), the minimum required sample size was obtained as 224 samples. Finally by considering the 20% attrition and for increasing the power of the study as well, 270 patients were included in the study. This calculated minimum sample size affords over 80% statistical power in our study based on the command “power oneproportion” in Stata software version 14.10.

Patients and sample collection

Diagnostic confirmation of β-thalassemia major

All patients enrolled in this study had a well-established, medically confirmed diagnosis of β-thalassemia major. This study was deliberately limited to patients with β-thalassemia major, the most severe form of thalassemia, characterized by lifelong transfusion dependency, iron overload, iron-chelation therapy, frequent hospitalizations, and a high rate of splenectomy. These factors are associated with significant immune dysfunction and an increased susceptibility to opportunistic fungal infections. Other thalassemia phenotypes differ substantially in clinical severity, transfusion requirements, immune status, and healthcare exposure. Inclusion of these heterogeneous groups could have introduced confounding variability; therefore, restricting the cohort to β-thalassemia major ensured population homogeneity and strengthened the internal validity of the study. Diagnosis was verified through a multi-faceted approach combining clinical history and definitive laboratory criteria, consistent with national and international guidelines for the management of thalassemia [35]. Confirmation was based on three criteria including clinical diagnosis, hematological confirmation, and molecular analysis. Diagnosis was confirmed through a review of comprehensive medical records from the Thalassemia Research Center, documenting a history of transfusion-dependent anemia from early childhood and the classic clinical features of undertreated β-thalassemia major, including growth retardation, hepatosplenomegaly, and skeletal deformities, such as thalassemia facies, genu valgum, and evidence of extramedullary hematopoiesis. For hematological confirmation; Transfusion-dependent thalassemia is characterised by reduced haemoglobin level (< 7 g/dL), MCV > 50 fL and < 70 fL and MCH > 12 and < 20 pg. The peripheral blood film in affected individuals demonstrates red blood cell morphological changes with microcytosis, hypochromia, anisocytosis, poikilocytosis and nucleated red blood cells (erythroblasts, NRBC). The number of erythroblasts is related to the degree of anaemia and is markedly increased after splenectomy. Finally, considering molecular analysis, common β-globin gene mutations are detected using PCR-based methods, typically reverse dot blot analysis or primer-specific amplification with probes/primers designed for the prevalent mutations in the patient’s population.

A thorough oral examination was conducted on all participants. Two oral swab specimens were collected using a sterile swab to gently rub the gum surface, teeth, dorsum of the tongue, hard palate, and cheek mucosa, as previously described [34]. The swabs were immediately placed in sterile containers and transported under refrigeration (4 °C) to the Invasive Fungi Research Center laboratory. All samples were processed within 2 h of collection [36]. Upon receipt, each swab was suspended in 1 mL of sterile distilled water, vortexed for 30 s, and serially diluted (1:1 and 1:10) in sterile distilled water. Subsequently, 0.1 mL of each dilution was inoculated onto CHROMagar Candida (CHROMagar Company, Paris, France) and spread evenly. Plates were incubated at 37 °C and examined at 48 and 72 h. The second swab was placed on a sterile slide, to which a drop of 10% potassium hydroxide (KOH) was added for microscopic examination to check for the presence of pseudohyphae and yeast cell forms. The colonies on the CHROM agar were initially identified based on the color they produced, following the manufacturer’s guidelines. Oral Candida colonization was identified in asymptomatic individuals without any lesions who had Candida-positive saliva or dorsal tongue swab cultures. The number of colony-forming units (CFU) per millilitre of saliva was counted by visual inspection and confirmed by two researchers, and finally, a value of 400 CFU/ml of Candida in saliva was established as the threshold [37]. In contrast, oral candidiasis was defined by the presence of clinically suspicious lesions, which may include inflammation, erythema, hyperplasia, pseudomembranes, or white plaques, along with positive saliva swab and culture results [38, 39].

Fungal isolates/phenotypic and genotypic characterization

The phenol–chloroform method was used to extract genomic DNA [40]. Briefly, the colonies were lysed using 300 µL of lysis buffer (composed of 200 mmol/L Tris–HCl (pH 7.5), 25 mmol/L EDTA, 0.5% (w/v) SDS, and 250 mmol/L NaCl). The mixture was incubated at 100 °C for 15 min, followed by centrifugation. The supernatant was combined with 20 µL of 3 M sodium acetate, then frozen at -20 °C for one hour and then centrifuged at 12,000 g for 10 min. The supernatants were mixed with an equal volume of ice-cold isopropanol and then centrifuged at 10,000 g for 10 min to precipitate the DNA. Following this, the pellet was washed with ice-cold 70% ethanol, air-dried, and resuspended in 50 µL of TE buffer, and the DNA was stored at -20 °C prior to use. The ITS1-5.8 S-ITS2 region was amplified using the universal primers ITS1 (5-TCCGTAGGTGAACCTGCGG-3) and ITS4 (5-TCCTCCGCTTATTGATATGC-3). The reaction was conducted in a final volume of 25 µL, which included 2 µL of extracted DNA, 0.4 mM of dNTPs, 1.5 mM of MgCl2, 30 pmol of each primer, 1.25 U of Taq DNA polymerase, and 2.5 µL of 10× PCR buffer. The PCR program was set as follows: an initial cycle at 95 °C for 5 min, followed by 30 cycles consisting of 1 min at 94 °C, 45 s at 55 °C, and 45 s at 72 °C, concluding with a final extension step at 72 °C for 7 min. Subsequently, the PCR products were digested using 1 U of the restriction enzyme MspI (1µL) in a final reaction volume of 15 µL, which included 3.5 µL of water, 1.5 µL of buffer, and 10 µL of the PCR product, incubated at 37 °C for 3 h. PCR amplicons and RFLP products were analyzed using agarose gel electrophoresis, with the PCR products loaded onto a 1.5% gel and the RFLP products on a 2% gel [41]. The products were stained with SYBR Safe DNA gel stain at a dilution of 1:10,000 in Tris/Borate/EDTA and subsequently photographed. Although sequencing is considered the gold standard for species identification, RFLP analysis allows for a rapid differentiation of Candida species and can also facilitate the detection of co-infections in cases where sequencing results are inconclusive or non-explanatory [42–44]. Furthermore, all Candida species that produced white, pink, red, or purple colonies on CHROMagar Candida and could not be identified at the species level using PCR-RFLP were subjected to further analysis. This involved the use of specific primers for C. auris, as previously described by Kordalewska et al., which yielded a 163-bp PCR product [45]. In addition, sequencing was performed for some Candida species and non-Candida yeast-like fungi, which were not distinguishable using PCR-RFLP. The internal transcribed spacer region was sequenced using primers ITS1 and ITS4 with SeqStudio Genetic Analyzer System (Thermo Fisher Scientific, MA), and finally, a comparative sequence analysis was done using the National Center for Biotechnology Information, Bethesda, MD (http://www.ncbi.nlm.nih.gov).

Antifungal susceptibility test

In vitro antifungal susceptibility testing was performed according to Clinical and Laboratory Standards Institute (CLSI) documents M27-A4 and M60 guidelines [46, 47]. Minimum inhibitory concentrations (MICs) were determined for eight antifungal agents, including luliconazole (Nihon Nohyaku Co), itraconazole (Janssen Research Foundation, Beerse, Belgium), fluconazole (Sigma-Aldrich, St. Louis, USA), anidulafungin (Cayman Chemical, USA), ketoconazole (Merck, Germany), voriconazole (Pfizer Sandwich Laboratories, UK), miconazole (Sigma-Aldrich, St. Louis, USA) and nystatin (Sigma-Aldrich, St. Louis, USA). The drugs were diluted in standard RPMI 1640 medium (Sigma Chemical Co.), which was buffered to pH 7.0 using 0.165 M 3-(N-Morpholino) propane sulfonic acid (MOPS, Sigma Chemical Co.), supplemented with L-glutamine and prepared without bicarbonate. This process resulted in the preparation of the drugs at two-fold their initial concentrations. The wells contained antifungal drugs at final concentrations ranging from 0.016 to 16 µg/mL for voriconazole, itraconazole, ketoconazole, miconazole, luliconazole, and nystatin, 0.064–64 µg/mL for fluconazole, and 0.008–8 µg/mL for anidulafungin. A suspension of fresh yeast colonies was prepared in a sterile saline solution, achieving a transmittance range of 75–77%. A working suspension was then created by performing a 1:100 dilution, resulting in a final concentration of 0.5 to 2.5 × 10³ CFU/mL. The microdilution plates were incubated at 35 °C, and the minimum inhibitory concentrations (MICs) were visually determined as the lowest concentration that completely inhibited growth for each drug after 24 h. Candida krusei (ATCC 6258) and Candida parapsilosis (ATCC 22019) were used as quality control strains to ensure the accuracy and reliability of the susceptibility testing results. All antifungal susceptibility tests were performed in duplicate. MICs were interpreted using species-specific CLSI breakpoints (M27-A4/M60) [48], categorizing isolates as susceptible, intermediate, or resistant. For agents without formal breakpoints (e.g., luliconazole, ketoconazole, nystatin), MIC distributions and geometric means were analyzed comparatively against published epidemiological cutoff values (ECVs) or relevant literature to infer susceptibility trends [49].

Statistical analysis

The mean value differences were analyzed using Student’s t-test with the SPSS statistical package (version 20.0). P values less than 0.05 were regarded as statistically significant.

Results

Clinical Features

Of the 270 patients diagnosed with β-Thalassemia major were recruited in this study. 52.96% (n = 143) of the study population were males and 47.03% were females (n = 127), with the mean ages ranging from 8 to 74 years (Table 1). Of 270 patients, 6.29% (n = 17) were positive for oral candidiasis (Fig. 1), and 8.14% (n = 22) were positive for Candida species colonization. Additionally, 85.5% (n = 231) were negative for direct examination and culture. In patients with oral candidiasis, the most frequently observed species were Candida albicans (52.38%; n = 11), Pichia kudriavzevii (formerly Candida krusei) (38.09%; n = 8), and Nakaseomyces glabratus (formerly Candida glabrata) (9.52%; n = 2). Notably, in the oral Candida infections group, alongside 21 Candida strains, one isolate was Cryptococcus diffluens recovered from the oral cavity. Among patients with Candida colonization, the most common species were Candida albicans (n = 23, 92%) and P. kudriavzevii (n = 2, 8%). In the current study, Candida auris was not detected in the populations examined. In addition, the nucleotide sequence data reported in our study are available in GenBank under accession numbers PV259180-PV259190.

Table 1.

Demographic and clinical data of patients with β-thalassemia major who participated in the present study

Variable	No. of patients with Oral candidiasis	No. of patients with Candida Colonization	No. of Total patients
Age range	23–55	31–60	8–74
Sex
Male	9	14	143
Female	8	8	127
Splenectomy
Yes	13	15	155
No	4	7	115
Receive iron-chelating drugs
Yes	16	22	264
No	1	0	6
Receive antibiotic drugs
Yes	11	15	147
No	6	7	123
Diabetes
Yes	3	2	23
No	14	20	247
Total	17	22	270

Fig. 1.

Direct smear of the Oral Candidiasis

In vitro susceptibility testing analysis

According to the CLSI susceptibility testing methodology documents, we analyzed the MIC₅₀, MIC₉₀, geometric mean MIC, mode, and MIC ranges. Table 2 presents the in vitro antifungal susceptibility profiles of the tested drugs against the isolated Candida strains. Among the Candida strains isolated from patients diagnosed with oral candidiasis infection, voriconazole and ketoconazole exhibited the lowest GM MIC values, indicating the most antifungal activity against all Candida strains followed by luliconazole (0.03 µg/mL), anidulafungin (0.04 µg/mL), miconazole (0.05 µg/mL), itraconazole (0.15 µg/mL), fluconazole (1.03 µg/mL), and nystatin (3.41 µg/mL). Regarding the two isolates of N. glabratus, voriconazole, ketoconazole, and luliconazole exhibited similar MICs of 0.016 µg/ml. In contrast, the MICs for other antifungal agents were as follows: itraconazole had an MIC of 0.25 µg/ml, miconazole ranged from 0.016 to 0.5 µg/ml, fluconazole ranged from 0.5 to 8 µg/ml, anidulafungin ranged from 0.008 to 1 µg/ml, and nystatin ranged from 0.5 to 16 µg/ml. Among the Candida strains isolated from oral colonization, voriconazole and ketoconazole demonstrated the lowest GM MIC (0.02 µg/mL) followed by miconazole (0.04 µg/mL), luliconazole (0.06 µg/mL), anidulafungin (0.09 µg/mL), itraconazole (0.15 µg/mL), fluconazole (0.92 µg/mL), and nystatin (2.29 µg/mL). Regarding the two strains of P. kudriavzevii isolated from the oral colonization group, voriconazole exhibited MICs ranging from 0.016 to 0.25 µg/mL. In comparison, the MICs for other antifungal agents were as follows: itraconazole ranged from 0.125 to 1 µg/mL, ketoconazole ranged from 0.016 to 0.5 µg/mL, luliconazole ranged from 0.063 to 0.5 µg/mL, miconazole ranged from 0.032 to 4 µg/mL, fluconazole ranged from 1 to 64 µg/mL, anidulafungin ranged from 0.063 to 4 µg/mL, and nystatin had an MIC of 2 µg/mL (Table 2).

Table 2.

Antifungal susceptibility profile of Candida species isolated from oral Candida infection and colonization among patients with β-thalassemia major

Fungal isolate Antifungal agents		Oral candidiasis group (n = 17)
		MIC (µg/ml)														MIC parameter (µg/ml)
		0.008	0.016	0.032	0.064	0.125	0.25	0.5	1	2	4	8	16	32	64	MIC50	MIC90	Range	GM	Mode
C. albicans (n = 11)	LUL		5	2	1		3									0.032	0.25	0.016–0.25	0.041	0.016
	ITC			2	1	4	3		1							0.125	0.25	0.032-1	0.133	0.125
	KTC		8	1	1	1										0.016	0.064	0.016–0.125	0.023	0.016
	VRC		9	1	1											0.016	0.032	0.016–0.064	0.019	0.016
	MCN		9			1			1							0.016	0.125	0.016-1	0.028	0.016
	FLC					1	2	6	1		1					0.5	1	0.125-4	0.5	0.5
	AFG	4	2	2	1		1		1							0.016	0.25	0.008-1	0.029	0.008
	NYT							1	3	4		1	2			2	16	0.5–16	2.416	2
P. kudriavzevii (n = 8)	LUL		7								1					0.016	0.016	0.016-4	0.031	0.016
	ITC			2		1	3		2							0.25	1	0.032-1	0.193	0.25
	KTC		6	1				1								0.016	0.032	0.016-0.5	0.026	0.016
	VRC		6	1			1									0.016	0.032	0.016–0.25	0.024	0.016
	MCN		2	2	1			1	1			1				0.032	1	0.016-8	0.126	0.032
	FLC						2	1	1	1	1	1		1		1	8	0.25-32	1.681	0.25
	AFG	4		1		1	1				1					0.032	0.25	0.008-4	0.044	0.008
	NYT								1		2	3	2			8	16	1–16	6.168	8
N. glabratus (n = 2)	LUL		2													-	-	0.016	0.016	0.016
	ITC					2										-	-	0.25	0.25	0.25
	KTC		2													-	-	0.016	0.016	0.016
	VRC		2													--	-	0.016	0.016	0.016
	MCN		1					1								-	-	0.016-0.5	0.089	-
	FLC							1				1				-	-	0.5-8	2	-
	AFG	1							1							-	-	0.008-1	0.089	-
	NYT							1					1			-	-	0.5–16	2.82	-
All Candida species (n = 21)	LUL		14	2	1		3				1					0.016	0.25	0 016 − 4	0.035	0.016
	ITC			4	1	5	8		3							0.25	1	0.032-1	0.163	0.25
	KTC		16	2	1	1		1								0.016	0.064	0.016-0.5	0.023	0.016
	VRC		17	2	1		1									0.016	0.032	0.016–0.25	0.020	0.016
	MCN		12	2	1	1		2	2			1				0.016	1	0.016-8	0.055	0.016
	FLC					1	4	8	2	1	2	2		1		0.5	8	0.125-32	0.905	0.5
	AFG	9	2	3	1	1	2		2		1					0.016	1	0.008-4	0.038	0.008
	NYT							2	4	4	2	4	5			4	16	0.5–16	3.50	16

	Oral colonization group (n = 22)
	MIC (µg/ml)															MIC parameter (µg/ml)
		0.008	0.016	0.032	0.064	0.125	0.25	0.5	1	2	4	8	16	32	64	MIC50	MIC90	Range	GM	Mode
C. albicans (n = 23)	LUL		8	4	2	2	5		1				1			0.032	0.25	0.016-16	0.054	0.016
	ITC		1	2	3	8	8						1			0.125	0.25	0.016-16	0.145	0.125
	KTC		20	2									1			0.016	0.032	0.016-16	0.022	0.016
	VRC		20	2									1			0.016	0.032	0.016-16	0.022	0.016
	MCN		12	5	1	3	1						1			0.016	0.125	0.016-16	0.039	0.016
	FLC						4	8	9	1					1	0.5	1	0.25-64	0.762	1
	AFG	2	3	3	2	8	3	1				1				0.125	0.25	0.008-8	0.082	0.125
	NYT								2	14	7					2	4	1–4	2.325	2
P. kudriavzevii (n = 2)	LUL				1			1								-	-	0.064-0.5	0.178	-
	ITC					1			1							-	-	0.125-1	0.353	-
	KTC		1					1								-	-	0.016-0.5	0.089	-
	VRC		1				1									-	-	0.016–0.25	0.063	-
	MCN			1							1					-	-	0.032-4	0.357	-
	FLC				1				1							-	-	0.064-1	8	-
	AFG				1						1					-	-	0.064-4	0.505	-
	NYT									2						-	-	2		2
All Candida species(n = 25)	LUL		8	4	3	2	5	1	1				1			0.064	0.25	0.016-16	0.061	0.016
	ITC		1	2	3	9	8		1				1			0.125	0.25	0.016-16	0.156	0.125
	KTC		21	2				1					1			0.016	0.032	0.016-16	0.025	0.016
	VRC		21	2			1						1			0.016	0.032	0.016-16	0.024	0.016
	MCN		12	6	1	3	1				1		1			0.032	0.25	0.016-16	0.046	0.016
	FLC						4	8	10	1					2	1	2	0.25-64	0.920	1
	AFG	2	3	3	3	8	3	1			1	1				0.125	0.5	0.008-8	0.095	0.125
	NYT								2	16	7					2	4	1–4	2.297	2

Abbreviations: LUL Luliconazole, ITC Itraconazole, KTC Ketoconazole, VRC Voriconazole, MCN Miconazole, FLC Fluconazole, AFG Anidulafungin, NYT Nystatin, GM Geometric mean

Applying CLSI interpretive criteria, the categorical susceptibility profiles of the isolated Candida species were determined (Summarized in Table 3). Among C. albicans isolates (n = 34), 94.2% (32/34) were susceptible, 2.9% (1/34) were intermediate, and 2.9% (1/34) were resistant to fluconazole. Notably, 97.1% (33/34) of C. albicans isolates were susceptible to voriconazole, with a single isolate (2.9%) classified as resistant. For anidulafungin, 91.2% (31/34) of C. albicans isolates were susceptible, 2.9% (1/34) were intermediate, and 5.9% (2/34) were resistant. All P. kudriavzevii isolates (n = 10) were susceptible to voriconazole. Regarding anidulafungin, 80% (8/10) of P. kudriavzevii isolates were susceptible, while 20% (2/10) were resistant. Both N. glabratus isolates (n = 2) were susceptible to fluconazole. For anidulafungin, one isolate (50%) was susceptible and the other (50%) was resistant. Among all Candida species (n = 46), 73.9% (34/46) were susceptible to fluconazole, with one isolate each (2.2%) classified as intermediate and resistant. Voriconazole susceptibility was observed in 93.5% (43/46) of isolates, with one resistant isolate (2.2%). Anidulafungin susceptibility was 86.9% (40/46), with 2.2% (1/46) intermediate and 10.9% (5/46) resistant isolates.

Table 3.

Clinical breakpoints (CBPs) and Epidemiological Cut-Off (ECV) values based on MICs for all Candida species as endorsed by CLSI

Species (No. of isolates)	Antifungal Agent	Susceptible, n (%)	Intermediate/Susceptible-Dose Dependent, n (%)	Resistant, n (%)	Wild Type, n (%)	Non-Wild Type, n (%)
C. albicans (n = 34)	VRC	33 (97.1)	0 (0)	1 (2.9)	-	-
	ITC	-	-	-	22 (64.7)	12 (35.3)
	FLC	32 (94.2)	1 (2.9)	1 (2.9)	-	-
	AFG	31 (91.2)	1 (2.9)	2 (5.9)	-	-
P. kudriavzevii (n = 10)	VRC	10 (100)	0 (0)	0 (0)	-	-
	ITC	-	-	-	10 (100)	0 (0)
	FLC	-	-	-	10 (100)	0 (0)
	AFG	8 (80)	0 (0)	2 (20)
N. glabratus (n = 2)	VRC	-	-	-	2 (100)	0 (0)
	ITC	-	-	-	2 (100)	0 (0)
	FLC	2 (100)	0 (0)	0 (0)	-	-
	AFG	1 (50)	0 (0)	1 (50)	-	-
All Candida spp. (n = 46)	VRC	43 (93.5)	0 (0)	1 (2.2)	2 (4.3)	0 (0)
	ITC	-	-	-	34 (73.9)	12 (26.1)
	FLC	34 (73.9)	1 (2.2)	1 (2.2)	10 (21.7)	0 (0)
	AFG	40 (86.9)	1 (2.2)	5 (10.9)	-	-

Discussion

β-thalassemia represents a significant population of patients who experience various hematological, endocrine, and immunological disorders. Infections are a common complication in patients with thalassemia and are the second leading cause of death after heart failure [1]. Predisposing factors for infections in thalassaemic patients include severe anaemia, iron overload, splenectomy, and a range of immune abnormalities, such as impairment of chemotaxis and phagocytosis of polymorphonuclear leukocytes and macrophages against microorganisms, low volume of T-helpers (CD₄), and an increase in T-suppressors (CD₈) and reduced levels of complement components (C3 and C4) [8, 14]. Candida species are the primary yeast present in the oral cavity. In healthy individuals, a balance is maintained among the various microorganisms in the oral flora. However, this balance can be disrupted by certain diseases, leading to an overgrowth of yeast species, which may then act as pathogens [16, 50]. The current study aimed to evaluate the prevalence of oral Candida colonization and infections in patients with β-thalassemia major. Additionally, we sought to screen for C. auris isolates, analyze species distribution, and assess the antifungal susceptibility profiles of the obtained isolates. Our findings revealed that out of the 270 patients, 6.29% (n = 17) tested positive for oral candidiasis, while 22 patients (8.14%) showed colonization by Candida species.

In addition, the demographic and clinical profile of our study (Table 1) offers further insight into the epidemiology of oral Candida in patients with β-thalassemia major. A notable observation is the relatively low overall prevalence of Candida isolation (14.43% combined colonization and infection) compared to some studies involving other immunocompromised cohorts [13, 51–53]. This discrepancy may be partially explained by specific clinical management factors within our patient group. For instance, a high percentage of enrolled patients (97.8%, 264/270) were receiving iron-chelating therapy. Effective iron chelation mitigates iron overload, a critical factor known to impair immune cell function (e.g., neutrophil and macrophage activity) and provide a crucial nutrient for fungal growth [54, 55]. Therefore, the widespread use of chelation therapy in our study likely represents a significant protective factor against fungal overgrowth, potentially contributing to the lower observed prevalence. Furthermore, the distribution of positive cases of Candida infection and colonization showed a higher number among splenectomized patients (28 out of 39 positive cases vs. 11 out of 39 non-splenectomized). The spleen is a key organ in innate and adaptive immunity, with a vital role in defending against encapsulated bacteria, fungi, and other pathogens circulating in the bloodstream [56]. Splenectomy is associated with long-term alterations in humoral immunity, particularly in IgM production and response to encapsulated organisms, which may extend vulnerability to other infections [57]. However, mucosal defense against Candida species depends more on epithelial barriers, neutrophil function, and Th17 responses [58, 59]. This may explain why, despite splenectomy, the prevalence of oral candidiasis (6.29%) in thalassemia patients is still lower than in other patients with primary neutrophil deficiency or other immunodeficiency disorders. Like Abdulaziz et al. study [34], interestingly, the sex distribution of Candida positivity (17 males vs. 16 females) was nearly equal, suggesting that sex is not a predominant risk factor within the context of the profound immune and metabolic dysregulation imposed by thalassemia major and its treatment.

Shaiegan et al. demonstrated that chemotactic migration and the ability to kill C. albicans were impaired in patients with thalassemia [32]. The research conducted by Al Wahab et al. showed a higher prevalence of C. albicans colonization in patients with thalassemia major compared to those with thalassemia minor and healthy controls [13]. The study also highlighted that iron overload, a consequence of frequent blood transfusions, leads to immune dysfunction, which increases the risk of oral fungal infections in patients with β-thalassemia [13]. In our study, C. albicans was the most commonly isolated strain from oral candidiasis and Candida colonization groups (52.38%, 92%, respectively). The finding that C. albicans was the most frequently isolated species from the oral cavities of healthy and diseased populations was expected, as this species is often reported as the predominant isolate in numerous studies [14, 31, 33, 34, 60–62]. In some studies, investigating the prevalence of Candida species in the oral cavities of patients with β-thalassemia, C. albicans has been recognized as the most prevalent species [14, 31, 33, 34]. Our results depicted that non-albicans Candida species (NAC) were more prevalent in the oral candidiasis group (47.61%) compared to the Candida colonization group (8%). Pichia kudriavzevii was the most frequently identified non-albicans Candida species in the oral candidiasis and the Candida colonization group, representing 38.09% and 8%, respectively. In a study examining Candida species isolated from patients with thalassemia major, C. albicans was found in 69.2% of cases, while non-albicans Candida species accounted for 30.8%. Among the non-albicans Candida isolates, 23% were identified as Candida tropicalis and 7.8% as P. kudriavzevii [33]. While C. albicans remains the most prevalent yeast associated with oral cavity infections, non-albicans Candida species (NCACs) have become increasingly linked to these infections, especially in older individuals, immunosuppressed patients, or those with prolonged exposure to antimicrobial agents [63]. In our study, C. auris was not recovered from any of the samples. Alfaifi et al. conducted a single-centre, retrospective institutional study to determine whether C. auris is a commensal colonizer of the oral cavity in patients with clinical evidence of oral candidiasis. In their study, C. auris was not recovered from any of the collected samples [64]. In another study, Pathirana et al. found that C. auris is highly susceptible to the host-produced salivary antimicrobial peptide histatin-5, a key component of innate immunity that plays a crucial role in its defence mechanisms [65]. Although C. auris was not detected in the oral cavity of patients with β-thalassemia major in our study, it is recommended that healthcare facilities establish surveillance protocols that include testing of oral samples, in addition to the existing screening from other sites. It is also important to remain vigilant for the potential colonization of C. auris on the oral mucosa, as this could potentially serve as a gateway for systemic infection [64, 66]. In the present study, antifungal susceptibility tests were conducted using the CLSI method for all isolates of Candida species from both the oral colonization and candidiasis groups. Overall, voriconazole (MIC₉₀: 0.032 µg/mL, GM-MIC: 0.02 µg/mL) and ketoconazole (MIC₉₀: 0.063 µg/mL, GM-MIC: 0.02 µg/mL) demonstrated the highest efficacy, while nystatin (MIC₉₀: 16 µg/mL, GM-MIC: 3.41 µg/mL) and fluconazole (MIC₉₀: 8 µg/mL, GM-MIC: 1.03 µg/mL) exhibited the weakest antifungal activity against all Candida species isolated from oral candidiasis group. In colonization group also, voriconazole (MIC₉₀: 0.032 µg/mL, GM-MIC: 0.02 µg/mL) and ketoconazole (MIC₉₀: 0.032 µg/mL, GM-MIC: 0.02 µg/mL had the highest antifungal activity and nystatin (MIC₉₀: 4 µg/mL, GM-MIC: 2.29 µg/mL) and fluconazole (MIC₉₀: 2 µg/mL, GM-MIC: 0.92 µg/mL) had the lowest antifungal activity against all isolated Candida species. Our findings indicate that different Candida species display varying levels of susceptibility to fluconazole, with C. albicans generally being more susceptible than other species such as N. glabratus and P. kudriavzevii. These results are consistent with Patel’s study on the susceptibility of Candida species in the oral cavities of patients with type 2 diabetes, which revealed that resistance to fluconazole was significantly higher among the non-albicans Candida isolates [62]. Abdulaziz et al. found that 85% of Candida species isolated from the oral cavities of patients with β-thalassemia major were resistant to fluconazole. This finding is consistent with the results of our study [34]. Concerning nystatin, research conducted by El Kasem et al. on the antifungal susceptibility of oral Candida species extracted from patients with thalassemia major revealed that 89.7% of all isolates were resistant to nystatin. This result also aligns with our findings [33]. Additionally, another study examining the antifungal susceptibility of Candida species isolated from cancer patients with oral lesions undergoing chemotherapy found that 100% of C. albicans isolates exhibited resistance to nystatin [67].

The present study focused exclusively on patients with β-thalassemia major, a subgroup with profound immunological vulnerability and a well-recognized predisposition to infectious complications. While this targeted approach strengthens the internal validity of the study and allows for a clearer interpretation of Candida epidemiology in a high-risk population, it may limit the direct generalizability of the findings to patients with other forms of thalassemia. Patients with thalassemia minor or non-transfusion-dependent thalassemia typically experience milder disease courses, lower iron burden, reduced exposure to transfusions and chelation therapy, and fewer immune alterations [68]. As a result, the prevalence of oral Candida colonization and infection, as well as antifungal susceptibility patterns, may differ substantially in these populations. Nevertheless, the current findings provide important insight into the burden and characteristics of oral Candida infections in the most clinically severe form of thalassemia. These data may serve as a reference framework for future comparative and multicenter studies designed to evaluate fungal epidemiology across different thalassemia phenotypes and to better define phenotype-specific risk factors and management strategies.

Conclusion

This study characterizes the oral Candida burden in β-thalassemia major, identifying 6.29% candidiasis and 8.14% colonization. The substantial proportion of non-albicans species, notably P. kudriavzevii (38.1% of infections), signals a critical etiological shift. Antifungal susceptibility data reveal superior in vitro activity of voriconazole compared to fluconazole and nystatin, challenging the empirical use of the latter, especially given the involvement of resistant non-albicans species. These findings necessitate a shift toward diagnosis-directed therapy, incorporating routine species identification and susceptibility testing. Despite the absence of C. auris, the prevalent immunomodulatory factors in this cohort, splenectomy, antibiotic use, and iron chelation, define a distinct risk profile requiring ongoing vigilance. We recommend structured oral surveillance and a diagnostic approach combining clinical and microbiological assessment. Future longitudinal studies are needed to clarify risk factor correlations and monitor resistance trends in this population.

Abbreviations

NTDT

Non-Transfusion-Dependent Thalassemia

TDT

Transfusion-Dependent Thalassemia

WHO

World Health Organization

KOH

Potassium Hydroxide

CFU

Colony-Forming Units

MIC

Minimum Inhibitory Concentrations

NAC

Non-albicans Candida Species

Authors’ contributions

I.H., conceptualization, methodology, project administration, supervision, writing-review and editing, writing-original draft, investigation, validation, Funding acquisition, visualization; Z.H., Z.Y., and J.J., writing-original draft, methodology, validation, writing-review and editing; M.A., M.T.H., S.R.A., and M.M., writing-original draft, validation, writing-review and editing; H.J., L.D., and H.K., writing-original draft, methodology, visualization, and writing-review and editing; M.N., and H.B., writing-original draft, writing-review and editing, supervision; E.N., software, formal analysis, and data curation. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Mazandaran University of Medical Sciences [grant number 19837].

Data availability

The data supporting this study’s findings are available from the corresponding author upon reasonable request.

Declarations

Ethics approval and consent to participate

The study was conducted in accordance with the Declaration of Helsinki and received supervision and approval from the research ethics committee of Mazandaran University of Medical Sciences in Sari, Iran (IR.MAZUMS.REC.1403.035). Moreover, the study’s objectives were clearly explained to the participants, and all patients provided written informed consent.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.