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

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

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).

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

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 Iron overload in beta-thalassemia major generates reactive oxygen species (ROS), impairing neutrophil and macrophage function, thus weakening immune defenses against Candida albicans.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

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.

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.