Oral Microbiome Diversity in Transfusion-Dependent Thalassemia Using a Metagenomic Approach in Indonesian Communities

Abstract

Beta-thalassemia major is an inherited disorder that requires lifelong blood transfusions, with the risk of complications including poor oral health and dental caries. The objective of this study was to compare the oral microbiome diversity and composition in transfusion-dependent thalassemia patients and relate it to oral hygiene and dental caries. A cross-sectional analysis of 35 patients of beta-thalassemia major aged 6–18 years was performed. The status of oral hygiene was examined through the Oral Hygiene Index—Simplified (OHI-S) and Decayed, Missing, and Filled Teeth (DMFT) index. Saliva was taken for DNA extraction, followed by the 16S rRNA sequencing of V3-V4 hypervariable regions. The bioinformatics pipeline in QIIME2 was utilized for analyzing the comparison of microbial composition and diversity in groups of varying oral hygiene status and severity of caries. Metagenomic analysis revealed 3334 Amplicon Sequence Variants (ASVs), of which the most prevalent genera were Streptococcus, Haemophilus, Veillonella, Rothia, and Prevotella. High-oral-hygiene groups presented increased levels of cariogenic bacteria, while moderate-oral-hygiene groups presented an equilibrated microbiome. No statistically significant differences in microbial diversity were found between the study groups (p > 0.05). This study sheds light on the critical importance of oral hygiene in microbiome diversity in patients with beta-thalassemia major.

1. Introduction

Thalassemia is a genetic disorder caused by mutations in genes coding for the globin chains in hemoglobin. It is one of the most common genetic diseases globally, with an estimated 7% carrier rate worldwide, equivalent to some 300,000–500,000 affected births per year [1]. Southeast Asian nations such as Indonesia, Malaysia, Thailand, Singapore, the Philippines, and Vietnam exhibit a high prevalence of beta-thalassemia, with carrier rates ranging from 3% to 10% of the population [2,3].

Beta-thalassemia major, the most common severe thalassemia, is due to a lack or restriction of hemoglobin synthesis that results in manifestations like anemia, iron overload, and growth and developmental impairment. Patients need to receive regular blood transfusions in order to survive, and the transfusions themselves lead to excess iron deposition and oxidative stress, as well as possible cellular and tissue damage [4,5]. The oral cavity is one of the regions that can be impacted by beta-thalassemia major. Research has indicated that patients suffering from the condition are prone to higher caries indices, gingivitis inflammation, and heightened vulnerability to infections caused by microbes in the oral region [6,7]. Despite these results, the general understanding of the oral microbiome in beta-thalassemia major patients is poor, particularly regarding the interconnection between oral hygiene and the risk of dental caries [8].

It was found in previous research that the rate of salivary flow, saliva composition, and oral hygiene could affect microbial colonization in thalassemia patients. Iron deposition within the gastrointestinal and oral cavity environment is also said to contribute to affecting bacterial growth in such patients. Research conducted among sickle cell anemia patients indicated that the disruption of immune system function and increased iron levels cause changes in the composition of the oral microbiome [9]. However, there has been limited in-depth research evaluating the oral microbiome of beta-thalassemia major patients, especially children with dental caries. To bridge this gap in knowledge, this study utilized metagenomic analysis to characterize the genomic content and microbial taxonomy of the oral microbiome in children with dental caries and beta-thalassemia major. A clearer understanding of this association may provide novel insights into the prevention and management of dental caries in this vulnerable patient population.

2. Materials and Methods

2.1. Ethical Approval

This study was conducted in strict adherence with the ethical standards established by the Institutional Review Board of Jenderal Soedirman University and in full compliance with the principles outlined in the Declaration of Helsinki. All research protocols involving human participants were thoroughly reviewed and approved by the Ethics Committee (approval number: 070/KEPK/PE/VIII/2024). All subjects or their legal guardians gave written informed consent prior to the collection of specimens.

2.2. Study Design and Sample Size

This cross-sectional exploratory study aimed to investigate microbial diversity and oral health status using samples collected from children diagnosed with beta-thalassemia major, in a peripheral region served by an Indonesian healthcare center [10]. The primary outcome of this study was microbial diversity and its association with dental caries and oral hygiene status.

Because this was an exploratory study, a formal sample size was not determined a priori; instead, we included all eligible and consenting children with beta-thalassemia major who presented at the health center during the study period. The final sample comprised 35 participants (mean age, 15.23 ± 2.04 years). Among these, 62.86% were classified as having poor oral hygiene, and 88.57% had dental caries. The mean DMFT score was 8.51 (±2.59), reflecting a very high dental caries index, while the mean OHI-S score was 3.60 (±0.98), indicating poor oral hygiene.

We acknowledge that the small sample size may limit the statistical power to detect significant differences in microbial diversity and composition between groups. Nevertheless, this study provides valuable preliminary data and highlights the need for future investigations with larger cohorts to validate these findings.

2.3. Clinical Examination and Sample Collection

Oral health was assessed by a trained dental clinician using the Oral Hygiene Index—Simplified (OHI-S) and the Decayed, Missing, and Filled Teeth (DMFT) index to determine dental hygiene and dental caries severity, respectively. Unstimulated saliva samples were then collected in sterile tubes following standard procedures and immediately stored at −80 °C until microbial genomic analysis.

2.4. Metagenomic Analysis

Genomic DNA was extracted using a commercial DNA extraction kit (zymoresearch, Irvine, CA, USA), as per the manufacturer’s instructions. Targeted primers were used to amplify V3–V4 hypervariable regions of the 16S rRNA gene of the bacterial genome and were then sequenced using a high-throughput shotgun Miseq sequencing platform (Illumina, Baltimore, MD, USA). All genome analysis was conducted at Genetica Science Laboratory, Jakarta, Indonesia.

The raw 16S rRNA gene sequences were processed using QIIME2 (v2023.1) pipeline. After demultiplexing and removing low-quality reads and chimeras with DADA2, Amplicon Sequence Variants (ASVs) were generated. Taxonomic assignment was performed against the SILVA 138 database with a 97% similarity threshold. To assess microbial diversity within samples (alpha diversity), we applied the Shannon index (to account for both richness and evenness) and the Simpson index (to reflect dominance within the microbial community). To account for variation in sequencing depth, rarefaction curves were constructed by repeatedly resampling sequences at different depths. The rarefaction curve reached a plateau at 10,000 reads per sample, indicating that the sequencing depth was sufficient to capture the microbial diversity present in all samples.

The normality of the data was first assessed by the Shapiro–Wilk test. To compare microbial diversity indices and microbial composition between groups, we used the Kruskal–Wallis test. A p-value of <0.05 was considered statistically significant for all comparisons.

3. Results

3.1. Participant Characteristics

A total of 35 participants were included in this study, consisting of 22 females (62.86%) and 13 males (37.14%) with a mean age of 15.23 years (SD ± 2.04). The majority of participants exhibited poor oral hygiene (62.86%), as reflected in a mean Oral Health Index—Simplified (OHIS) score of 3.60 (SD ± 0.98). Additionally, 88.57% of participants were categorized with a very high dental caries index, with a mean DMFT score of 8.51 (SD ± 2.59). Detailed demographic and clinical characteristics are presented in

Table 1. Characteristics of respondents.

Variable	Number
Age (years)	12–18
DMFT (caries index)
Very high	31 (88.57%)
High	4 (11.43%)
OHIS (oral hygiene)
Poor	22 (62.86%)
Medium	13 (37.14%)

.

3.2. Alpha Diversity and Rarefaction Analysis

To assess microbial richness and diversity within the groups, we evaluated the Observed ASVs, Shannon index, and Simpson index. The boxplot (Figure 1) shows the distribution of these diversity indices across the four groups. The Observed ASVs reflect microbial richness, while the Shannon and Simpson indices account for both the richness and evenness of microbial communities. Groups 1 and 2 demonstrated slightly higher microbial richness and diversity, as reflected by the greater Observed ASV and Shannon index values, although these differences were not statistically significant (p > 0.05). The Simpson index, which quantifies dominance within the community, remained relatively consistent across all groups, indicating a similar degree of microbial dominance and evenness. The rarefaction curve (Figure 2) illustrates that the number of observed species reaches a plateau at a sequencing depth of about 10,000 reads, suggesting that the sequencing effort was sufficient to capture the microbial diversity present in all samples. Each curve leveling off signals that additional sequencing would likely uncover few, if any, new ASVs, reflecting a comprehensive sampling of microbial communities in these groups.

The metagenomic analysis identified 3334 Amplicon Sequence Variants (ASVs) from the 35 saliva samples. Participants were categorized into four groups based on their dental caries index and oral hygiene status. Group 1 consisted of individuals with very high DMFT scores and poor oral hygiene, while Group 2 included those with very high DMFT scores but moderate oral hygiene. Group 3 comprised participants with high DMFT scores and poor oral hygiene, whereas Group 4 consisted of those with high DMFT scores and moderate oral hygiene.

3.3. Microbiome Diversity

The heatmap illustrates the microbial community structure at the genus level across all samples. Distinct clusters of microbial communities were observed, reflecting differences in microbial composition related to dental health and hygiene groups. Genera such as Streptococcus, Veillonella, and Rothia were predominantly abundant across all samples, while other taxa displayed variability in their relative abundance. The color gradients highlight these fluctuations, suggesting a potential association between microbial profiles and dental health conditions in beta-thalassemia major patients. The diagram in Figure 3 illustrates distinct microbial distribution patterns across the subjects.

The analysis of the top 10 bacterial taxa at the genus level across the four study groups revealed consistent microbial profiles with no statistically significant differences between groups (p > 0.05) (Figure 4). The groups were classified based on their dental caries index (DMFT) and oral hygiene status (OHI-S): Group 1, very high dental caries, poor oral hygiene; Group 2, very high dental caries, moderate oral hygiene; Group 3, high dental caries, poor oral hygiene; Group 4, high dental caries, moderate oral hygiene. Each color within the stack corresponds to a microbial genus, illustrating its relative abundance within the microbial community of each group. The microbial composition highlights differences in community structure related to dental health and hygiene conditions. As illustrated, Streptococcus was the predominant genus across all groups.

To assess whether the microbial diversity and composition were significantly different across dental health and hygiene groups, we performed Kruskal–Wallis tests. The p-values for these comparisons were all greater than 0.05, indicating that there were no statistically significant differences in microbial diversity or community composition (p > 0.05).

The concentric rings represent various taxonomic levels (phylum, class, order, family, genus), radiating outward from the center (Figure 5). The sizes of the segments reflect the relative prevalence of every microbial group within the community. The multilevel interactive visualization underscores the microbial diversity included in the oral microbiome and enables intense probing into its taxonomic structure. To see an interactive Krona chart, visit https://tinyurl.com/idkronamicro (we thank http://usegalaxy.fr for this online tool; URL accessed on 30 June 2025).

4. Discussion

This study demonstrates that children with beta-thalassemia major are at heightened risk for dental caries, reflected by a mean DMFT score of 8.51, significantly higher than that reported in general pediatric populations. We also report that poor oral hygiene was prevalent among participants, with 62.86% exhibiting a poor oral hygiene status as measured by an average OHIS score of 3.60.

This high caries index is indicative of severe oral health challenges faced by this population and underscores the need for targeted interventions. These findings align with [11,12], who observed a prevalence of dental caries in 56–86.7% of thalassemia patients with a mean DMFT of 4.2, highlighting the widespread nature of this issue across different cohorts. Comparatively, the DMFT scores in healthy pediatric populations tend to be substantially lower, underscoring the unique vulnerability of thalassemia patients to oral health deterioration [13].

These outcomes may be explained from a physiological perspective. The disease alters the salivary composition and reduces the salivary flow rate, weakening the natural protective mechanisms against caries [14]. Additionally, the craniofacial abnormalities frequently observed in thalassemia patients, such as maxillary protrusion and malocclusion, create anatomical challenges that trap food particles and hinder effective oral hygiene. These children also often experience delayed dental development and enamel defects, further predisposing them to caries formation [15,16].

Beyond the direct effects of the disease, its treatment exacerbates oral health challenges. Regular blood transfusions and iron chelation therapy can contribute to xerostomia (dry mouth), further compromising saliva’s protective function. Moreover, the primary focus on managing life-threatening complications often means that dental care becomes a lower priority for both families and healthcare providers. Frequent hospital visits and prolonged treatments can also disrupt regular dental care routines, leading to inconsistent oral hygiene practices [11].

Psychosocial factors further contribute to poor oral health outcomes in these patients. The physical burden of the disease, coupled with chronic fatigue, may reduce the child’s ability or motivation to maintain oral hygiene. Families managing a chronic illness may prioritize other medical expenses over dental care due to financial constraints or time limitations [9]. Additionally, a lack of awareness regarding the importance of oral health in thalassemia management among patients, caregivers, and even healthcare professionals can lead to neglect in preventive dental care. Together, these interconnected factors highlight the need for targeted interventions to improve oral health in children with beta-thalassemia major [17].

The markedly elevated caries index in thalassemia patients may be attributed to multiple interrelated factors, including an altered salivary composition, impaired oral immune defenses, and poor oral hygiene practices. Salivary changes in these patients, such as reduced flow rates, diminished buffering capacity, and altered biochemical composition, play a critical role in creating an environment conducive to caries development. Oxidative stress, a hallmark of beta-thalassemia, further exacerbates this issue by disrupting salivary gland function and impairing the protective properties of saliva [18]. These disruptions can lead to an increased susceptibility to dental caries, as the oral environment becomes more favorable to cariogenic bacteria and less capable of neutralizing acidic byproducts of bacterial metabolism [19].

Systemic factors such as chronic anemia and iron overload may indirectly influence oral health by weakening the overall immune system and increasing oxidative stress levels. This systemic oxidative imbalance can have local repercussions in the oral cavity, further aggravating the risk of caries. The combined effect of these systemic and local factors makes dental caries a significant and multifaceted issue in beta-thalassemia major, requiring a comprehensive approach that addresses both systemic health and localized oral care. Future studies should explore integrated strategies that include nutritional interventions, antioxidant therapies, and enhanced dental hygiene protocols to mitigate these risks effectively [15].

The analysis of the oral microbiome composition in different study groups revealed distinct variations in microbial diversity and abundance. The heatmap plot (Figure 3) and Krona diagram (Figure 5) illustrate the overall microbial distribution, with Streptococcus being the dominant genus, followed by other notable genera such as Haemophilus, Veillonella, Rothia, and Prevotella.

Streptococcus is a known commensal organism and constitutes a significant portion of the normal oral flora in healthy individuals. However, despite its status as a normal inhabitant, certain Streptococcus species can become opportunistic pathogens under favorable conditions, potentially exacerbating existing oral health issues [20]. The prevalence of Streptococcus, a known early colonizer in dental biofilms, suggests its crucial role in shaping the oral microbial environment, potentially influencing caries susceptibility [21]. Additionally, the presence of genera such as Veillonella and Prevotella, which are associated with anaerobic metabolism and acid production, could contribute to the progression of dental caries, particularly in individuals with poor oral hygiene.

The stacked bar plot (Figure 4) provides a comparative overview of the microbial community structure among the four study groups, categorized by their dental caries index and oral hygiene status. Notably, the relative abundance of Streptococcus and Veillonella remains consistently high across all groups, while variations in other taxa, such as Prevotella and Rothia, suggest potential differences in microbial resilience and adaptation to oral environmental changes. Group 1 and Group 3, characterized by poor oral hygiene, show higher proportions of caries-associated bacteria, reinforcing the link between microbial dysbiosis and oral disease severity. Meanwhile, Group 2 and Group 4, with moderate oral hygiene, exhibit slightly greater microbial diversity, which may contribute to a more balanced oral microbiome.

Although we did not find statistically significant differences in microbial diversity (both alpha and beta) across the dental health and hygiene groups, we did observe trends in microbial composition at the genus level. Certain genera, such as Streptococcus and Prevotella, appeared more abundant in groups with poor dental hygiene and dental caries. Nonetheless, these trends did not reach statistical significance, likely due to the small sample size and limited statistical power of the study. Therefore, we interpret these microbial patterns with caution and view them as preliminary observations that may guide future investigations. Large-scale, well-powered studies are needed to validate these trends and to clarify their potential role in dental disease pathogenesis in children with beta-thalassemia major.

We can further highlight the inter-individual variability in microbial abundance, emphasizing the differences in microbiome composition across participants. The distribution of bacterial genera across samples indicates that certain taxa exhibit a higher prevalence in specific individuals, suggesting the influence of host factors such as diet, salivary composition, and oral hygiene practices. The variation in microbial signatures underscores the complexity of oral ecosystem dynamics, reinforcing the need for targeted preventive strategies based on microbial profiling [22].

Overall, these findings highlight the significant influence of oral hygiene and dental health status on microbiome composition. The dominance of cariogenic bacteria in groups with poor oral hygiene suggests an increased risk for dental caries, whereas moderate oral hygiene appears to support a more diverse microbial community. This dysbiosis creates an acidic oral environment, which undermines enamel integrity and facilitates bacterial colonization, thus accelerating demineralization and elevating the risk of dental caries [23]. These insights underscore the importance of personalized oral healthcare strategies that consider microbial profiling to mitigate caries risk and promote oral health [24].

The implications for thalassemia management are profound. First, these findings underscore the necessity of integrating oral health monitoring into the routine care of patients with beta-thalassemia major. Regular dental check-ups can help identify and address microbial imbalances early, potentially mitigating their progression into more severe dental issues. Second, interventions aimed at restoring oral microbiome balance—such as probiotics, prebiotics, or microbiome-targeted therapies—may be particularly beneficial in reducing the prevalence of cariogenic species [25,26].

Educational programs aimed at improving oral hygiene behaviors among beta-thalassemia patients and their caregivers are essential [27]. Teaching effective brushing techniques, advocating for the use of dental floss, and encouraging the use of fluoride-containing toothpaste and mouthwashes could significantly reduce bacterial plaque accumulation and caries risk. These steps not only improve oral health outcomes but also have broader implications for systemic health, as poor oral health can exacerbate systemic inflammation and oxidative stress [28].

Collectively, these findings underscore the interplay between systemic oxidative stress, immune dysfunction, and oral microbial imbalances in children with beta-thalassemia major. Future studies should investigate targeted interventions aimed at reducing oxidative stress, optimizing salivary composition, and promoting better oral hygiene to mitigate dental caries and improve overall oral health in this vulnerable population.

Limitations

The collection and analysis of dental plaque or saliva samples for microbial studies, however, present several analytical and logistical challenges. These include variability in sampling techniques, storage conditions, and processing methods—all of which can influence microbial profiles and affect comparability across studies [29]. Furthermore, factors such as the site of sample collection, time of day, and patient-related variables (age, health status, medication use) can introduce biases in microbial community composition. To minimize these effects, we standardized sample collection protocols, maintained a consistent freezing and storage procedure at −80 °C, and applied rigorous laboratory controls during processing and sequencing. Nevertheless, future studies should aim to harmonize methods across different research groups to enable more accurate comparisons and to account for these logistical and analytical variables in their interpretations.

We must also state that the sample size was relatively small and may not fully represent the diversity of the dental microbial community in this population. A key limitation of this study was the lack of a healthy control group, making it difficult to directly compare microbial profiles and dental abnormalities in children with beta-thalassemia major with those without the condition. Additionally, we were unable to adjust for potential confounders such as dietary habits, oral hygiene practices, iron and ferritin levels, or transfusion frequency—all of which could significantly impact both microbial composition and dental health outcomes.

While 16S rRNA sequencing offers valuable insights into microbial taxonomy and relative abundance, its resolution is often insufficient to distinguish species-level differences or elucidate functional capabilities. As a result, this approach does not allow for the evaluation of microbial metabolic activity, virulence determinants, or antibiotic resistance markers—factors that could significantly influence the development of dental pathologies. To address these limitations, future studies could incorporate metatranscriptomic or shotgun metagenomic techniques, which would enable a more detailed assessment of microbial functionality. Such approaches may help clarify the specific mechanisms through which microbial communities contribute to dental abnormalities in pediatric patients with beta-thalassemia major.

5. Conclusions

This study shows no significant differences in oral microbiome composition across individuals with varying dental caries severity and oral hygiene status. Streptococcus was dominant, with poor oral hygiene linked to higher levels of potentially pathogenic genera like Prevotella and Rothia. Clinically, these findings suggest a need for integrated dental care strategies tailored to this population, including frequent dental check-up schedules, personalized oral hygiene instructions, topical fluoride applications, and dental sealants to help reduce dental caries risk. Dental practitioners should be vigilant regarding dental abnormalities and poor oral health in children with beta-thalassemia and collaborate closely with pediatricians and hematologists to implement a multidisciplinary approach to patient care.