Prevalence of <i>Candida albicans</i> in the oral cavity of Beta Thalassemia Major and Thalassemia Minor Patients

Saba Sami Abd Al Wahab; Maha Adel Mahmood

doi:10.12688/f1000research.162460.1

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Research Article

Prevalence of Candida albicans in the oral cavity of Beta Thalassemia Major and Thalassemia Minor Patients

[version 1; peer review: 2 approved]

Saba Sami Abd Al Wahab¹, Maha Adel Mahmood¹

PUBLISHED 03 Jun 2025

Author details Author details

¹ Department of Basic Sciences, College of Dentistry, Baghdad University, Baghdad, Baghdad Governorate, Iraq

Saba Sami Abd Al Wahab
Roles: Data Curation, Formal Analysis, Investigation, Methodology, Software, Writing – Original Draft Preparation

Maha Adel Mahmood
Roles: Conceptualization, Writing – Original Draft Preparation, Writing – Review & Editing

OPEN PEER REVIEW

REVIEWER STATUS

This article is included in the Pathogens gateway.

Abstract

Aims

To examine the correlation between iron, ferritin concentrations, and C. albicans infection in individuals with beta-thalassemia major and beta-thalassemia minor compared with healthy subjects.

Materials and methods

It involved 90 participants, thirty patients with thalassemia major and thirty patients with thalassemia minor compared with thirty healthy controls. Saliva samples were obtained and cultivated to isolate, identify, and calculate the viable colony count of C. albicans in (cfu/ml). In contrast, serum levels of iron and ferritin were quantified using chemical analyzers.

Result

Showed that 73.33% of thalassemia major group exhibited oral C. albicans colonization, which is significantly higher than that of thalassemia minor group 40% and control group 6.67%. Biochemical analyses revealed significantly higher iron 278.82 μg/dl and ferritin 2783.80 ng/ml levels in major group p<0.001 when compared with both thalassemia minor group 122.652 μg/dl, 74.723 ng/ml and control groups 127.438 μg/dl and 63.150 ng/ml respectively. C. albicans colony count in saliva was significantly elevated in beta-thalassemia major, as compared with beta-thalassemia minor group and control group.

Conclusion

Findings suggest that iron overload, which results from recurrent blood transfusions and causes immune dysfunction, contributes to higher risk of oral fungal infections in Beta-thalassemia patients compared with controls.

Keywords

Thalassemia Major, Thalassemia Minor, Candida albicans, Iron Overload, Ferritin levels

Corresponding author: Maha Adel Mahmood

Competing interests: No competing interests were disclosed.

Grant information: The author(s) declared that no grants were involved in supporting this work.

Copyright: © 2025 Abd Al Wahab SS and Mahmood MA. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

How to cite: Abd Al Wahab SS and Mahmood MA. Prevalence of Candida albicans in the oral cavity of Beta Thalassemia Major and Thalassemia Minor Patients [version 1; peer review: 2 approved]. F1000Research 2025, 14:555 (https://doi.org/10.12688/f1000research.162460.1) First published: 03 Jun 2025, 14:555 (https://doi.org/10.12688/f1000research.162460.1) Latest published: 21 Aug 2025, 14:555 (https://doi.org/10.12688/f1000research.162460.2)

Introduction

Thalassemia is a hereditary condition affecting hemoglobin, the condition involves a decrease or absence in the formation of one or more globin chains in the hemoglobin tetramers, resulting in uncontrolled destruction of red blood cells and, ultimately, severe anemia.^1,2 Beta-thalassemia arises from mutations that impact many stages of beta-globin protein synthesis, encompassing transcription, translation, and the stability of beta-globin production.³ One of the most prevalent genetic illnesses in humans is β-thalassemia. Around 60,000 infants worldwide are born with β-thalassemia annually, predominantly in countries of the Mediterranean but also in the Middle East, West Africa, India, and South-East Asia.⁴ The estimated prevalence of thalassemia carriers is 3 to 10 individuals per 100 individuals. Given a population of 200 million individuals, a birth rate of 20%, and a thalassemia carrier rate of around five percent, it is projected that there will be an annual occurrence of 2,500 kids born with thalassemia congenital illness.⁵ Patients with β-thalassemia often have blood transfusions and suffer from iron overload.⁶ Serum ferritin (SF) indicates probable iron excess.⁷ Serum ferritin is an effective monitoring instrument for iron overload in thalassemia major.⁸ In healthy individuals, the average serum ferritin (SF) levels range from 12 to 300 μg/L for men and from 12 to 150 μg/L for females.⁹ Serum ferritin levels exceeding 1000 μg/L are indicative of iron overload and are linked to adverse outcomes, including increased mortality and organ injury, a more significant likelihood of cardiac events, and hepatic difficulties.¹⁰ Candida albicans is a fungal infection that results in approximately 1.7 million fatalities annually on a global scale, primarily affecting those with weakened immune systems and several underlying medical problems.¹¹ The prevalence of infectious illnesses by Candida has consistently risen since the 1970s, potentially attributed to an elevated susceptibility to opportunistic infections, advancements in clinical techniques for detecting fungi-related hospital-acquired infections, and the emergence of antifungal resistance resulting from prolonged treatment exposure.^12,13 The clinical features of oral cavities in patients with thalassemia include pointed and shortened root morphology, taurodontism, and a characteristic chipmunk appearance. These individuals often have a higher caries index and display gingival hypertrophy, which suggests gingival inflammation. Additionally, these patients are susceptible to infections caused by bacteria or fungi, including C. albicans.¹⁴ Thalassemia, especially thalassemia major, is linked to a heightened incidence of C. albicans colonization in the oral cavity. This increased vulnerability is chiefly attributable to variables including iron accumulation from repeated blood transfusions, which creates a nutrient-rich environment favorable for fungal proliferation, and immunological impairment caused by the disease and its therapies.¹⁵ This study aims to examine the correlation between iron and ferritin levels and C. albicans distribution in individuals with major and minor beta-thalassemia compared with healthy controls.

Methods

Subject selection

The saliva and blood samples were collected between December 2023 and March 2024 from a total of sixty patients; thirty of them were individuals diagnosed with Beta Thalassemia Major, and thirty were diagnosed with Beta Thalassemia Minor attending the Thalassemia Centers of Ebn-Albalidy and Al-Karama hospitals in Baghdad compared with thirty apparently healthy individuals served as control. Data collected from each participant included demographic information (name, age, gender) and duration of illness. The Research Ethics Committee of the University of Baghdad College of Dentistry granted ethical permission for the study, with reference number 889 and project number 889824, on January 11, 2024. The research was conducted in conformity with the principles established in the Declaration of Helsinki. Before doing the analysis, all patient data was anonymized.

Inclusion criteria

Inclusion criteria included patients with stable Beta Thalassemia Major (T Major) without other diseases or medication influences this group representing the 1^st patients group, and stable Beta Thalassemia minor (T Minor) patients with no significant health changes, representing the 2^nd patients group compared with apparently healthy individuals with no blood disorders or chronic illnesses affecting iron metabolism representing the control group. All participants with ages ranged between 18-60 years.

Exclusion criteria

Exclusion criteria included any subject between 18 and 60 years of age but with an inability to provide informed consent or with conditions or treatments that could affect the biomarkers being studied. Indeed, pregnant or lactation women were also excluded.

Serum and saliva collection

Three milliliters of unstimulated saliva were obtained from each participant by the spitting method,¹⁶ and subsequently transferred to sterile tubes. The samples were centrifuged at 4000 rpm for 3 minutes to separate cellular debris. For blood samples, 5 mL were obtained through venipuncture into sterile tubes, centrifuged for 15 minutes at 3000 rpm, and the serum extracted using an automated pipette. Both serum and saliva were preserved at -20°C until later usage.

Isolation and counting of Candida albicans

Saliva samples from both patients and control groups were processed using a serial dilution method¹⁷ 4.5 ml of nutrient broth was added to sterile tubes, and 0.5 ml of freshly collected saliva was introduced into the first tube, followed by inoculating the following tubes successively to achieve the tenfold dilutions. One hundred microliters of each dilution were distributed over a plate of Sabouraud Dextrose Agar (SDA) medium containing 1% chloramphenicol and incubated at 37°C for 24-48 hours. The resulting colony count were determined by CFU/ml.

Microscopic identification of Candida albicans

C. albicans were initially identified and explored microscopically to observe the oval or elongated yeast cells exhibiting budding and pseudohyphae. Additionally, the C. albicans inoculum was incubated in 3-5 ml of human serum. A small quantity of the grown serum was placed on a glass slide and examined under a microscope to determine the presence or absence of germ tube formation.

Identification by using chromogenic test and VITEK- 2 compact system

C. albicans were subcultured onto a chromogenic medium (CHRO Magar TM Candida media) and then incubated at 37°C for 48 to 72 hours. This technique uses substrates linked to chemical dyes to distinguish several species of Candida by the coloration of the developing colonies. For additional identification, the VITEK^® 2 COMPACT was employed to accurately identify C. albicans isolates to the species level, according to the manufacturer’s guidelines (Biomerieux/France).

Measurement of serum iron and ferritin levels by chemistry analyzers

The method employs microparticle-enhanced immunoturbidimetry using a Thermo Scientific™ Indiko™ Clinical Chemistry Analyze (Finland). Sera samples from patients and controls were utilized and processed with microparticles coated with rabbit antibodies specific to either human ferritin or iron. The rabbit antibodies specific to human ferritin and iron were not sourced directly from live animal cells in our laboratory. They were obtained externally as part of commercial reagent kits from Thermo Scientific: the Human Ferritin Chemistry Analyzer Kit (Ref 981949, 4 × 4 mL, Indiko™) and the Human Iron Chemistry Analyzer Kit (Ref 981236, 10 × 20 mL, Konelab™, Indiko™). These kits provide pre-coated microparticles with polyclonal rabbit anti-human antibodies, validated for use with the Thermo Scientific™ Indiko™ Clinical Chemistry Analyzer. The measurement of immunocomplex formation for Ferritin is conducted by assessing absorbance variations at 700 nm, within a detection range of 0.32–20 ng/mL, iron immunocomplex formation is quantified by absorbance at 600 nm.

Statistical analysis

The statistical analyses were carried out by applying the SPSS 15 software (SPSS Inc., IL, USA, https://spssdownload.com/spss-software-version-15-free-download). It was employed the Shapiro-Wilk test, Kruskal-Wallis H test, and the Pairwise test. The p-value of less than 0.05 was considered statistically significant.

Result

The research study examined the age-based disparities in mean values for the T Major and T Minor groups and for the control group. T Minor group demonstrated a superior mean value of age (35.533 ± 7.523) years, followed by the T Major group (23.233 ± 7.398) years in comparison with the control group as it had a somewhat elevated mean than the minor group (29.667 ± 9.517). Table 1.

Table 1. Age-specific distribution of mean ± standard deviation values among groups.

	Control		T Major		T Minor
	N	Mean ± S.D.	N	Mean± S.D.	N	Mean ± S.D.
Total	30	29.667 ± 9.517	30	23.233 ± 7.398	30	35.533 ± 7.523

Isolation and identification of Candida albicans

C. albicans isolates were isolated and diagnosed successfully from the saliva of patients suffering from B-thalassemia. C. albicans display distinctive colonies. When cultivated on (SDA), the colonies exhibited a smooth, creamy texture and were white to off-white in hue. They are often grown with a delicate texture. Over time, the colonies may display the development of a corrugated surface Figure 1. CHRO Magar was utilized for more accurate identification. In this instance, colonies of C. albicans display a characteristic green coloration due to the enzymatic activity of the yeast. The combination of these characteristics with microscopic identification facilitates the accurate diagnosis of C. albicans infections.

Figure 1. (A) Shows colonies of C. albicans on SDA (B) Shows C. albicans on CHRO Magar.

For advanced identification, all isolates were accurately identified using the VITEK 2 Compact System, with a probability of 88% to 90%, hence demonstrating the system’s efficiency in identifying C. albicans. Figure 2 and Table 2 show the distribution of C. albicans among the groups as the T Major has the highest carriage rate, as twenty-two out of thirty patients have C. albicans accounting for (73.33%) of the group. In contrast, in the T Minor, only twelve out of thirty patients carrying C. albicans represented (40%) of the group, and the control group exhibited the lowest prevalence rate with merely two individuals, constituting (6.67%) of the healthy subjects. The results indicated a markedly elevated colonization rate of C. albicans in the major group relative to the minor and control groups, respectively.

Figure 2. The distribution of salivary C. albicans among groups.

Table 2. Distribution of C. albicans among groups.

	T Major		T Minor		Control
	Present (%)	Absent	Present	Absent	Present	Absent
No. Total	22(73.33)	8	12(40)	18	2(6.67)	28

Measurement of Iron and ferritin levels in B-thalassemia patients

This study analyzed two biochemical parameters, Iron and ferritin, across the beta thalassemic Major and Minor patients in comparison with the control group. Descriptive statistics, comprising the median, which was computed for each parameter simultaneously with C. albicans. Statistical analysis indicated substantial differences across the groups (p < 0.001) for all parameters, as the T-Major group revealed a higher iron, ferritin, and C. albicans count when compared with the results of both T Minor and control groups, as seen in Table 3.

Table 3. Descriptive statistics for iron, ferritin, and C. albicans levels among study groups.

Parameters	Groups	Descriptive statistics
Parameters	Groups	Median	X²	P-value
Iron (μg/dl)	T Major	270.016	58.919	0.000
	T Minor	117.599
	Control	116.985
Ferritin (mg/ml)	T Major	2783.800	59.376	0.000
	T Minor	77.800
	Control	63.150
C. albicans (x10⁴) cfu/ml	T Major	1.715	28.068	0.000
	T Minor	0
	Control	0

The T Major group for Iron demonstrated the most excellent median (270.016) in comparison to the Control (116.985) and T Minor (117.599). Likewise, for Ferritin, T Major had a significantly higher median (2783.800), greatly surpassing T Minor (77.800) and control (63.150). In addition to these biochemical markers, C. albicans levels in saliva were significantly elevated in the T Major group (1.715 x10⁴ cfu/ml) compared to the Minor and control as the median has zero value for both groups. These data demonstrate substantial increases in these parameters within the T Major group vs. other groups.

The results in Table 4 illustrated the pairwise analysis of Iron, Ferritin, and C. albicans levels among the T Major, T Minor and control groups demonstrated substantial increases in the T Major group relative to the control group for all three parameters (p < 0.001). Likewise, significant differences were noted between the T Major and T Minor groups across all parameters (p ≤ 0.005), further emphasizing the heightened levels in the T Major group. No significant differences were observed between the T Minor and control groups for the biochemical parameters, Iron and Ferritin (p = 0.748 and p = 0.851, respectively), and the difference for C. albicans was also not statistically significant between these groups (p = 0.106).

Table 4. Pairwise comparisons and P-values for iron, ferritin, and Candida albicans concentrations across study cohorts.

Parameters	Groups		Pairwise test	p-value
Iron (μg/dl)	Control	T Major	-43.717	0.000
	Control	T Minor	2.167	0.748
	T Major	T Minor	45.883	0.000
Ferritin (mg/ml)	Control	T Major	-44.367	0.000
	Control	T Minor	1.267	0.851
	T Major	T Minor	45.633	0.000
C. albicans	Control	T Major	-31.433	0.000
	Control	T Minor	-12.567	0.106
	T Major	T Minor	18.867	0.005

Correlation between markers for study groups

The results in Tables 5, 6, and 7 showed no significant correlations among all markers under investigation (iron, ferritin, and C. albicans levels) in both patient groups (T Major and T Minor) and the control group, except for a significant correlation noted between iron and ferritin levels in the control group.

Table 5. Correlation between markers in T Major group.

Parameters		C. albicans	Ferritin
Iron (μg/dl)	r	-0.062	0.092
Iron (μg/dl)	p	0.746	0.628
Ferritin (mg/ml)	r	-0.214
Ferritin (mg/ml)	p	0.257

Table 6. Correlation between markers in the T Minor group.

Parameters		C. albicans	Ferritin
Iron (μg/dl)	r	0.082	0.343
Iron (μg/dl)	p	0.668	0.064
Ferritin (mg/ml)	r	0.037
Ferritin (mg/ml)	p	0.844

Table 7. Correlation between markers in the control group.

Parameters		C. albicans	Ferritin
Iron (μg/dl)	r	0.040	0.499
Iron (μg/dl)	p	0.833	0.005
Ferritin (mg/ml)	r	0.065
Ferritin (mg/ml)	p	0.734

Discussion

The result of this study indicated that 73.33% of the significant thalassemia patient group had oral cavity colonization by C. albicans, a substantially greater rate compared to the minor group at 40% when compared with the control group which displayed the lowest prevalence at 6.67%. A markedly elevated mean candidal count was noted in thalassemic patients in comparison to the healthy cohort, the findings closely agree with another study conducted in Jordan, which indicated that Candida species were identified in 74% of thalassemic patients. Similarly, the present results obtained were comparable to another study conducted in Egypt, as C. albicans was isolated in 69.2% of cases, whereas 30.8% were non- C. albicans.¹⁸ The data obtained exhibited a higher elevation rate of C. albicans when compared with a local study performed in Najaf, Iraq, which evaluated samples from 50 thalassemia patients, of which only 14(28%) demonstrated positive growth for Candida species.¹⁹ One of the primary reasons that significant C. albicans have infected some minor patients is the immune problems that may have increased sensitivity to oral fungal colonization in these thalassemic patients.²⁰ This fact may be due to the deficiency in neutrophils and macrophage chemotaxis, which have been documented in thalassemic individuals, especially in those experiencing iron overload.²¹ Anemia may serve as a predisposing factor for oral fungal colonization in people with thalassemia.²² Another reason is that repeated blood transfusions lead to iron accumulation in salivary glands, resulting in the development of non-transferrin-bound iron (NTBI), which circulates in plasma and generates reactive oxygen species (ROS).²³ Iron accumulation in salivary gland acini cells induces inflammation and reduces salivary output and components.²⁴ Salivary components bolster the immune system’s response to infections caused by C. albicans by preventing the spread and protecting the mucosal epithelial barrier. Reduced salivary production in beta-thalassemia major patients can lead to dysbiosis, causing C. albicans to overgrow and adhere to the oral epithelium.²⁵ Increased levels of iron and ferritin create an environment favorable for the growth of C. albicans in the major group.²⁶ The findings of the current study reveal a unique biochemical and microbiological profile in the T Major group, marked by considerably increased levels of iron, ferritin, and C. albicans as compared to the T Minor and control groups. The T Major group exhibited significantly elevated iron and ferritin levels, indicating modified iron metabolism, potentially associated with inflammation or stress responses, alongside markedly increased C. albicans concentrations, indicating possible microbiome disruption or immune system changes. The pairwise analysis confirmed that these elevations were statistically significant (p < 0.001), differentiating the T Major group, from each of minor and control group. Still, no significant changes were noted between the T Minor and control groups for iron and ferritin (p = 0.748 and p = 0.851) or C. albicans (p = 0.106). The results obtained correspond with another two previous studies, one conducted in Turkey and the other in Indonesia. Both studies investigated iron and ferritin levels in saliva and serum across various groups, including thalassemia patients (major and minor) and controls. The findings of these studies indicate significantly elevated blood ferritin and iron levels in the significant thalassemia group compared to both minor and control groups.^27,28 The findings of the current investigation align with those of a study performed in Pakistan involving 155 patients with T Major. The study also revealed markedly increased ferritin and iron levels in the T Major group relative to the control group.²⁹ The increased concentrations of iron and ferritin in individuals in the T Major group are mainly due to the frequent blood transfusions necessary for the treatment of severe anemia. Each transfusion sends roughly 200 mg of iron into the body,³⁰ and due to the absence of a natural physiological mechanism for excreting excess iron, patients progressively collect iron.³¹ This iron overload results in the accumulation of surplus iron in tissues and organs, evidenced by markedly increased serum ferritin levels, a crucial indicator of iron storage.³² Moreover, the inadequate erythropoiesis in beta-thalassemia significantly leads to heightened intestinal iron absorption, hence intensifying iron buildup. In the absence of sufficient chelation therapy, iron excess may escalate to dangerous levels, leading to problems such as organ damage.³³ Conversely, beta-thalassemia minor is marked by less severe anemia as it resulting from a singular faulty beta-globin gene.³⁴ Individuals with this illness generally do not necessitate blood transfusions, hence reducing the risk of iron excess from exogenous sources.³⁵ Moreover, the extent of inefficient erythropoiesis is considerably reduced in beta-thalassemia minor, resulting in a better-controlled iron absorption and storage mechanism. As a result, their iron and ferritin levels typically stay within normal limits.³⁶ The present work illustrated a correlation between biochemical markers and colonization by Candida albicans in thalassemia patients. The increased levels of C. albicans in individuals in the T Major group may be indirectly associated with the raised concentrations of iron and ferritin in this population.²¹ Iron is essential for microbial proliferation and pathogenicity, particularly in fungal infections such as C. albicans.³⁷ In instances of iron overload, such as in beta-thalassemia major, excess free iron in the bloodstream and tissues could be served as a plentiful substrate for Candida, facilitating its growth.³⁸ Ferritin, a principal iron storage protein, is also raised in beta-thalassemia major as a result of continuous transfusions and poor erythropoiesis.^39,40 Candida might have evolved ways to extract iron from host ferritin, hence augmenting its survival and proliferation in iron-abundant settings. Conversely, patients with beta-thalassemia minor demonstrate normal or nearly normal iron and ferritin levels owing to the lack of recurrent transfusions and modest inefficient erythropoiesis.⁴¹ As a result, there is an absence of surplus iron to promote Candida proliferation, and their immune systems are often less impaired. This leads to markedly reduced amounts of C. albicans in patients with beta-thalassemia minor relative to those with beta-thalassemia major.⁴²

Conclusion

This study shows the heightened prevalence of C. albicans colonization in patients with thalassemia major relative to individuals with thalassemia minor and healthy controls. The results indicate that iron overload, diminished immune system impairment, and contribute to the increased vulnerability to fungal infections in thalassemia major patients. These findings underscore the significance of iron control and iron chelation therapy in averting problems, such as oral fungal infections, and indicate that consistent monitoring may enhance patient outcomes.

Ethics and consent

The Research Ethics Committee of the University of Baghdad College of Dentistry granted ethical permission for the study, with reference number 889 and project number 889824, on January 11, 2024. The study started on February 1, 2024, and was conducted in conformity with principles established in the Declaration of Helsinki. Before doing the analysis, all patient data was anonymized.

All participants provided written informed consent, as approved by the Research Ethics Committee of the University of Baghdad, College of Dentistry, which granted ethical permission for the study. The patients were fully informed about the study’s objectives, procedures, and potential benefits, and were assured of their complete freedom to participate. Consent was obtained for the collection of both blood and saliva samples.

Data availability

Underlying data

Figshare: my data.xlsx. https://doi.org/10.6084/m9.figshare.28399424.v1.⁴³

The project contains the following underlying data:

• my data.xlsx

Data are available under the terms of the Creative Commons Attribution International license (CC BY 4.0).

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Author details Author details

¹ Department of Basic Sciences, College of Dentistry, Baghdad University, Baghdad, Baghdad Governorate, Iraq

Saba Sami Abd Al Wahab
Roles: Data Curation, Formal Analysis, Investigation, Methodology, Software, Writing – Original Draft Preparation

Maha Adel Mahmood
Roles: Conceptualization, Writing – Original Draft Preparation, Writing – Review & Editing

Competing interests

No competing interests were disclosed.

Grant information

The author(s) declared that no grants were involved in supporting this work.

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Published: 21 Aug 2025, 14:555

https://doi.org/10.12688/f1000research.162460.2

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https://doi.org/10.12688/f1000research.162460.1

© 2025 Abd Al Wahab SS and Mahmood MA. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Abd Al Wahab SS and Mahmood MA. Prevalence of Candida albicans in the oral cavity of Beta Thalassemia Major and Thalassemia Minor Patients [version 1; peer review: 2 approved]. F1000Research 2025, 14:555 (https://doi.org/10.12688/f1000research.162460.1)

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Reviewer Report 13 Aug 2025

Pradana Zaky Romadhon, Airlangga University, Surabaya, Indonesia

Approved

https://doi.org/10.5256/f1000research.178665.r392347

The submitted article is quite interesting and has been written with good structure and clarity. However, there are several point that might enhance the article, which we outline as follows:

Candida albicans levels, which were

The submitted article is quite interesting and has been written with good structure and clarity. However, there are several point that might enhance the article, which we outline as follows:

Candida albicans levels, which were also assessed as quantitative data, are not described in the Methods section. The authors should include a clear description of the methodology used to obtain quantitative data for C. albicans.
In the Discussion section, a brief explanation should be provided regarding the mechanism by which iron overload impairs the immune system, making thalassemia patients more susceptible to Candida albicans infections.
The authors are also encouraged to elaborate on the limitations that may have contributed to the lack of significant correlation among the investigated markers (iron, ferritin, and C. albicans levels) across the T major, T minor, and control groups.

Is the work clearly and accurately presented and does it cite the current literature?

Yes
Is the study design appropriate and is the work technically sound?

Yes
Are sufficient details of methods and analysis provided to allow replication by others?

Partly
If applicable, is the statistical analysis and its interpretation appropriate?

I cannot comment. A qualified statistician is required.
Are all the source data underlying the results available to ensure full reproducibility?

Yes
Are the conclusions drawn adequately supported by the results?

Yes

Competing Interests: No competing interests were disclosed.

Reviewer Expertise: Thalasemia and Hemophilia

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

CITE

Report a concern

Author Response 10 Sep 2025

Saba Abd Al Wahab, Department of Basic Sciences, College of Dentistry, Baghdad University, Baghdad, Iraq

10 Sep 2025

Author Response

We sincerely thank the reviewer for their positive feedback on the structure and clarity of our article and for their valuable suggestion to enhance the manuscript.

Question: Candida albicans ... Continue reading We sincerely thank the reviewer for their positive feedback on the structure and clarity of our article and for their valuable suggestion to enhance the manuscript.

Question: Candida albicans levels, which were also assessed as quantitative data, are not described in the Methods section. The authors should include a clear description of the methodology used to obtain quantitative data for C. albicans
Response: We appreciate the reviewer’s question. The quantitative assessment of Candida albicans levels was performed by determining the viable colony count in colony-forming units per milliliter (cfu/ml).

Question: In the Discussion section, a brief explanation should be provided regarding the mechanism by which iron overload impairs the immune system, making thalassemia patients more susceptible to Candida albicans infections.
Response: We appreciate the reviewer’s thoughtful suggestion to include a brief explanation of the mechanism by which iron overload impairs the immune system, increasing susceptibility to Candida albicans infections in thalassemia patients. Additional information has been added to the revised manuscript

Question The authors are also encouraged to elaborate on the limitations that may have contributed to the lack of significant correlation among the investigated markers (iron, ferritin, and C. albicans levels) across the T major, T minor, and control groups.

Response: We thank the reviewer for their valuable suggestion to elaborate on the limitations that may have contributed to the lack of significant correlation among iron, ferritin, and Candida albicans levels across the T Major, T Minor, and control groups. To address this, we propose adding the following paragraph at the end of the Discussion section
(The current study has a limitation that may explain the absence of significant correlations between iron, ferritin, and Candida albicans levels across the study. The relatively small sample size (n=30 per group) may have limited statistical power to detect weaker correlations. Additionally, variability in transfusion frequency, chelation therapy, and individual immune responses among T Major patients could have confounded the relationship between iron, ferritin, and fungal levels).
We sincerely thank the reviewer for their positive feedback on the structure and clarity of our article and for their valuable suggestion to enhance the manuscript.

Question: Candida albicans levels, which were also assessed as quantitative data, are not described in the Methods section. The authors should include a clear description of the methodology used to obtain quantitative data for C. albicans
Response: We appreciate the reviewer’s question. The quantitative assessment of Candida albicans levels was performed by determining the viable colony count in colony-forming units per milliliter (cfu/ml).

Question: In the Discussion section, a brief explanation should be provided regarding the mechanism by which iron overload impairs the immune system, making thalassemia patients more susceptible to Candida albicans infections.
Response: We appreciate the reviewer’s thoughtful suggestion to include a brief explanation of the mechanism by which iron overload impairs the immune system, increasing susceptibility to Candida albicans infections in thalassemia patients. Additional information has been added to the revised manuscript

Question The authors are also encouraged to elaborate on the limitations that may have contributed to the lack of significant correlation among the investigated markers (iron, ferritin, and C. albicans levels) across the T major, T minor, and control groups.

Response: We thank the reviewer for their valuable suggestion to elaborate on the limitations that may have contributed to the lack of significant correlation among iron, ferritin, and Candida albicans levels across the T Major, T Minor, and control groups. To address this, we propose adding the following paragraph at the end of the Discussion section
(The current study has a limitation that may explain the absence of significant correlations between iron, ferritin, and Candida albicans levels across the study. The relatively small sample size (n=30 per group) may have limited statistical power to detect weaker correlations. Additionally, variability in transfusion frequency, chelation therapy, and individual immune responses among T Major patients could have confounded the relationship between iron, ferritin, and fungal levels).
Competing Interests: No competing interests were disclosed. Close
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COMMENTS ON THIS REPORT

Author Response 10 Sep 2025

Saba Abd Al Wahab, Department of Basic Sciences, College of Dentistry, Baghdad University, Baghdad, Iraq

10 Sep 2025

Author Response

We sincerely thank the reviewer for their positive feedback on the structure and clarity of our article and for their valuable suggestion to enhance the manuscript.

Question: Candida albicans ... Continue reading We sincerely thank the reviewer for their positive feedback on the structure and clarity of our article and for their valuable suggestion to enhance the manuscript.

Question: Candida albicans levels, which were also assessed as quantitative data, are not described in the Methods section. The authors should include a clear description of the methodology used to obtain quantitative data for C. albicans
Response: We appreciate the reviewer’s question. The quantitative assessment of Candida albicans levels was performed by determining the viable colony count in colony-forming units per milliliter (cfu/ml).

Question: In the Discussion section, a brief explanation should be provided regarding the mechanism by which iron overload impairs the immune system, making thalassemia patients more susceptible to Candida albicans infections.
Response: We appreciate the reviewer’s thoughtful suggestion to include a brief explanation of the mechanism by which iron overload impairs the immune system, increasing susceptibility to Candida albicans infections in thalassemia patients. Additional information has been added to the revised manuscript

Question The authors are also encouraged to elaborate on the limitations that may have contributed to the lack of significant correlation among the investigated markers (iron, ferritin, and C. albicans levels) across the T major, T minor, and control groups.

Response: We thank the reviewer for their valuable suggestion to elaborate on the limitations that may have contributed to the lack of significant correlation among iron, ferritin, and Candida albicans levels across the T Major, T Minor, and control groups. To address this, we propose adding the following paragraph at the end of the Discussion section
(The current study has a limitation that may explain the absence of significant correlations between iron, ferritin, and Candida albicans levels across the study. The relatively small sample size (n=30 per group) may have limited statistical power to detect weaker correlations. Additionally, variability in transfusion frequency, chelation therapy, and individual immune responses among T Major patients could have confounded the relationship between iron, ferritin, and fungal levels).
We sincerely thank the reviewer for their positive feedback on the structure and clarity of our article and for their valuable suggestion to enhance the manuscript.

Question: Candida albicans levels, which were also assessed as quantitative data, are not described in the Methods section. The authors should include a clear description of the methodology used to obtain quantitative data for C. albicans
Response: We appreciate the reviewer’s question. The quantitative assessment of Candida albicans levels was performed by determining the viable colony count in colony-forming units per milliliter (cfu/ml).

Question: In the Discussion section, a brief explanation should be provided regarding the mechanism by which iron overload impairs the immune system, making thalassemia patients more susceptible to Candida albicans infections.
Response: We appreciate the reviewer’s thoughtful suggestion to include a brief explanation of the mechanism by which iron overload impairs the immune system, increasing susceptibility to Candida albicans infections in thalassemia patients. Additional information has been added to the revised manuscript

Question The authors are also encouraged to elaborate on the limitations that may have contributed to the lack of significant correlation among the investigated markers (iron, ferritin, and C. albicans levels) across the T major, T minor, and control groups.

Response: We thank the reviewer for their valuable suggestion to elaborate on the limitations that may have contributed to the lack of significant correlation among iron, ferritin, and Candida albicans levels across the T Major, T Minor, and control groups. To address this, we propose adding the following paragraph at the end of the Discussion section
(The current study has a limitation that may explain the absence of significant correlations between iron, ferritin, and Candida albicans levels across the study. The relatively small sample size (n=30 per group) may have limited statistical power to detect weaker correlations. Additionally, variability in transfusion frequency, chelation therapy, and individual immune responses among T Major patients could have confounded the relationship between iron, ferritin, and fungal levels).
Competing Interests: No competing interests were disclosed. Close
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Reviewer Report 13 Jun 2025

Anwer Faisal, Mustansiriyah University, Baghdad, Iraq

Approved

https://doi.org/10.5256/f1000research.178665.r389913

This well-designed study provides valuable clinical insights into how iron overload may impact Candida infections in thalassemia patients. By comparing three distinct groups - those with major and minor forms of the disease alongside healthy controls - the researchers uncovered striking differences in fungal colonization rates that clearly track with disease severity. The finding that over 70% of major thalassemia patients carried oral Candida, compared to just 6% of healthy individuals, is particularly noteworthy for clinicians managing these cases.
While the laboratory methods appear sound and the statistical approach appropriate, we did note that the study could have been strengthened by including more details about how thalassemia was diagnosed in participants. A simple mention of whether genetic testing or hemoglobin electrophoresis was used would help readers better assess the accuracy of patient grouping.
Despite this minor limitation, the research makes a compelling case for the connection between iron overload and fungal susceptibility in this vulnerable population.

Is the work clearly and accurately presented and does it cite the current literature?

Yes
Is the study design appropriate and is the work technically sound?

Partly
Are sufficient details of methods and analysis provided to allow replication by others?

Yes
If applicable, is the statistical analysis and its interpretation appropriate?

Yes
Are all the source data underlying the results available to ensure full reproducibility?

Yes
Are the conclusions drawn adequately supported by the results?

Yes

Competing Interests: No competing interests were disclosed.

Reviewer Expertise: Microbiology - pathogenic bacteria

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

CITE

Report a concern

Author Response 10 Sep 2025

Saba Abd Al Wahab, Department of Basic Sciences, College of Dentistry, Baghdad University, Baghdad, Iraq

10 Sep 2025

Author Response

We thank the reviewer for their insightful feedback and for recognizing the study's compelling case for the connection between iron overload and fungal susceptibility in beta-thalassemia patients.

The diagnosis ... Continue reading We thank the reviewer for their insightful feedback and for recognizing the study's compelling case for the connection between iron overload and fungal susceptibility in beta-thalassemia patients.

The diagnosis of beta-thalassemia major and beta-thalassemia minor was confirmed at the Thalassemia Centers of Ebn-Albalidy and Al-Karama hospitals in Baghdad through a combination of clinical evaluation and laboratory testing. All participants underwent hemoglobin electrophoresis to assess hemoglobin patterns, which is a standard diagnostic tool for differentiating beta-thalassemia major
We thank the reviewer for their insightful feedback and for recognizing the study's compelling case for the connection between iron overload and fungal susceptibility in beta-thalassemia patients.

The diagnosis of beta-thalassemia major and beta-thalassemia minor was confirmed at the Thalassemia Centers of Ebn-Albalidy and Al-Karama hospitals in Baghdad through a combination of clinical evaluation and laboratory testing. All participants underwent hemoglobin electrophoresis to assess hemoglobin patterns, which is a standard diagnostic tool for differentiating beta-thalassemia major
Competing Interests: No competing interests were disclosed. Close
Report a concern
Respond or Comment

COMMENTS ON THIS REPORT

Author Response 10 Sep 2025

Saba Abd Al Wahab, Department of Basic Sciences, College of Dentistry, Baghdad University, Baghdad, Iraq

10 Sep 2025

Author Response

We thank the reviewer for their insightful feedback and for recognizing the study's compelling case for the connection between iron overload and fungal susceptibility in beta-thalassemia patients.

The diagnosis ... Continue reading We thank the reviewer for their insightful feedback and for recognizing the study's compelling case for the connection between iron overload and fungal susceptibility in beta-thalassemia patients.

The diagnosis of beta-thalassemia major and beta-thalassemia minor was confirmed at the Thalassemia Centers of Ebn-Albalidy and Al-Karama hospitals in Baghdad through a combination of clinical evaluation and laboratory testing. All participants underwent hemoglobin electrophoresis to assess hemoglobin patterns, which is a standard diagnostic tool for differentiating beta-thalassemia major
We thank the reviewer for their insightful feedback and for recognizing the study's compelling case for the connection between iron overload and fungal susceptibility in beta-thalassemia patients.

The diagnosis of beta-thalassemia major and beta-thalassemia minor was confirmed at the Thalassemia Centers of Ebn-Albalidy and Al-Karama hospitals in Baghdad through a combination of clinical evaluation and laboratory testing. All participants underwent hemoglobin electrophoresis to assess hemoglobin patterns, which is a standard diagnostic tool for differentiating beta-thalassemia major
Competing Interests: No competing interests were disclosed. Close
Report a concern

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Version 1 03 Jun 25	read	read

Anwer Faisal, Mustansiriyah University, Baghdad, Iraq
Pradana Zaky Romadhon, Airlangga University, Surabaya, Indonesia

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Reviewer Report

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22 Aug 2025 | for Version 2

Anwer Faisal, Mustansiriyah University, Baghdad, Iraq

1 Views Cite this report Responses(0)

Approved

I have reviewed the authors' revisions and the summary of changes made in response to the peer review reports. The authors have adequately addressed all the required modifications and concerns raised during the review process.
I confirm my Approved status for this revised version. The improvements made strengthen the manuscript and I believe it is now ready for indexing.

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

Microbiology - pathogenic bacteria

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

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Reviewer Report

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13 Aug 2025 | for Version 1

Pradana Zaky Romadhon, Airlangga University, Surabaya, Indonesia

3 Views Cite this report Responses(1)

Approved

The submitted article is quite interesting and has been written with good structure and clarity. However, there are several point that might enhance the article, which we outline as follows:

Candida albicans levels, which were also assessed as quantitative data, are not described in the Methods section. The authors should include a clear description of the methodology used to obtain quantitative data for C. albicans.
In the Discussion section, a brief explanation should be provided regarding the mechanism by which iron overload impairs the immune system, making thalassemia patients more susceptible to Candida albicans infections.
The authors are also encouraged to elaborate on the limitations that may have contributed to the lack of significant correlation among the investigated markers (iron, ferritin, and C. albicans levels) across the T major, T minor, and control groups.

Is the work clearly and accurately presented and does it cite the current literature?

Yes
Is the study design appropriate and is the work technically sound?

Yes
Are sufficient details of methods and analysis provided to allow replication by others?

Partly
If applicable, is the statistical analysis and its interpretation appropriate?

I cannot comment. A qualified statistician is required.
Are all the source data underlying the results available to ensure full reproducibility?

Yes
Are the conclusions drawn adequately supported by the results?

Yes

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

Thalasemia and Hemophilia

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

Respond to this report

Responses (1)

Author Response

10 Sep 2025

Saba Abd Al Wahab, Department of Basic Sciences, College of Dentistry, Baghdad University, Baghdad, Iraq

We sincerely thank the reviewer for their positive feedback on the structure and clarity of our article and for their valuable suggestion to enhance the manuscript.

Question: Candida albicans levels, which were also assessed as quantitative data, are not described in the Methods section. The authors should include a clear description of the methodology used to obtain quantitative data for C. albicans
Response: We appreciate the reviewer’s question. The quantitative assessment of Candida albicans levels was performed by determining the viable colony count in colony-forming units per milliliter (cfu/ml).

Question: In the Discussion section, a brief explanation should be provided regarding the mechanism by which iron overload impairs the immune system, making thalassemia patients more susceptible to Candida albicans infections.
Response: We appreciate the reviewer’s thoughtful suggestion to include a brief explanation of the mechanism by which iron overload impairs the immune system, increasing susceptibility to Candida albicans infections in thalassemia patients. Additional information has been added to the revised manuscript

Question The authors are also encouraged to elaborate on the limitations that may have contributed to the lack of significant correlation among the investigated markers (iron, ferritin, and C. albicans levels) across the T major, T minor, and control groups.

Response: We thank the reviewer for their valuable suggestion to elaborate on the limitations that may have contributed to the lack of significant correlation among iron, ferritin, and Candida albicans levels across the T Major, T Minor, and control groups. To address this, we propose adding the following paragraph at the end of the Discussion section
(The current study has a limitation that may explain the absence of significant correlations between iron, ferritin, and Candida albicans levels across the study. The relatively small sample size (n=30 per group) may have limited statistical power to detect weaker correlations. Additionally, variability in transfusion frequency, chelation therapy, and individual immune responses among T Major patients could have confounded the relationship between iron, ferritin, and fungal levels).

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Competing Interests

No competing interests were disclosed.

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Reviewer Report

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13 Jun 2025 | for Version 1

Anwer Faisal, Mustansiriyah University, Baghdad, Iraq

9 Views Cite this report Responses(1)

Approved

Is the work clearly and accurately presented and does it cite the current literature?

Yes
Is the study design appropriate and is the work technically sound?

Partly
Are sufficient details of methods and analysis provided to allow replication by others?

Yes
If applicable, is the statistical analysis and its interpretation appropriate?

Yes
Are all the source data underlying the results available to ensure full reproducibility?

Yes
Are the conclusions drawn adequately supported by the results?

Yes

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

Microbiology - pathogenic bacteria

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

Respond to this report

Responses (1)

Author Response

10 Sep 2025

Saba Abd Al Wahab, Department of Basic Sciences, College of Dentistry, Baghdad University, Baghdad, Iraq

We thank the reviewer for their insightful feedback and for recognizing the study's compelling case for the connection between iron overload and fungal susceptibility in beta-thalassemia patients.

The diagnosis of beta-thalassemia major and beta-thalassemia minor was confirmed at the Thalassemia Centers of Ebn-Albalidy and Al-Karama hospitals in Baghdad through a combination of clinical evaluation and laboratory testing. All participants underwent hemoglobin electrophoresis to assess hemoglobin patterns, which is a standard diagnostic tool for differentiating beta-thalassemia major

View more View less

Competing Interests

No competing interests were disclosed.

Alongside their report, reviewers assign a status to the article:

Approved - the paper is scientifically sound in its current form and only minor, if any, improvements are suggested

Approved with reservations - A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.

Not approved - fundamental flaws in the paper seriously undermine the findings and conclusions

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Prevalence of Candida albicans in the oral cavity of Beta Thalassemia Major and Thalassemia Minor Patients

Abstract

Aims

Materials and methods

Result

Conclusion

Keywords

Introduction

Methods

Subject selection

Inclusion criteria

Exclusion criteria

Serum and saliva collection

Isolation and counting of Candida albicans

Microscopic identification of Candida albicans

Identification by using chromogenic test and VITEK- 2 compact system

Measurement of serum iron and ferritin levels by chemistry analyzers

Statistical analysis

Result

Table 1. Age-specific distribution of mean ± standard deviation values among groups.

Isolation and identification of Candida albicans

Figure 1. (A) Shows colonies of C. albicans on SDA (B) Shows C. albicans on CHRO Magar.

Figure 2. The distribution of salivary C. albicans among groups.

Table 2. Distribution of C. albicans among groups.

Measurement of Iron and ferritin levels in B-thalassemia patients

Table 3. Descriptive statistics for iron, ferritin, and C. albicans levels among study groups.

Table 4. Pairwise comparisons and P-values for iron, ferritin, and Candida albicans concentrations across study cohorts.

Correlation between markers for study groups

Table 5. Correlation between markers in T Major group.

Table 6. Correlation between markers in the T Minor group.

Table 7. Correlation between markers in the control group.

Discussion

Conclusion

Ethics and consent

Data availability

Underlying data

References

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