Molecular insights into macrolide resistant Mycoplasma pneumoniae isolates from outpatient clinics in Tehran, Iran

Abstract

The rising incidence of macrolide-resistant Mycoplasma pneumoniae (MRMP) in Asia is a significant public health concern due to its implications in severe diseases. However, limited reliable data is available on this topic from Iran. To address this gap, we carried out a study in Tehran focusing on antimicrobial susceptibility and genotype distribution of MRMP. Following the CLSI M43-A guidelines, we assessed the growth inhibition of 54 M. pneumoniae isolates from outpatient clinics using four antimicrobial agents including azithromycin, erythromycin, clindamycin, and levofloxacin. In addition, Multilocus sequence typing (MLST) was performed on 20 randomly selected MRMP isolates. Our findings revealed alarming high resistance rates to erythromycin (85.1%) and azithromycin (85.1%), with significantly lower rates for clindamycin (9.25%). Notably, all isolates were susceptible to levofloxacin. Clonal complex analysis identified CC1 as the predominant complex (65% of isolates), with ST3 (50%) and ST14 (15%) being the most prevalent sequence types. This study provides novel insights into antimicrobial resistance and molecular epidemiology of M. pneumoniae in Iran. The high rates of MRMP isolates underscores the strong need for continued monitoring and antimicrobial stewardship of M. pneumoniae infections.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-026-35614-2.

Introduction

Mycoplasma pneumoniae is a common cause of respiratory diseases, including M. pneumoniae pneumonia (MPP), as well as a wide range of extrapulmonary systemic manifestations^1,2. Several European countries have reported the prevalence rates of 15–20% in adults and over 40% in children, particularly during epidemic cycles occurring every 3 to 7 years³. Although the pathogenesis of this pathogen remains unclear, recent evidence suggests it involves both direct effects and immune-mediated responses mechanisms contribute to its pathogenesis⁴. Despite the infection is typically self-limiting, recent reports indicated an increasing number of severe and life-threatening pneumonia cases⁵.

Recent investigations have highlighted aggravating factors of MPP severity, including aberrant immune responses, macrolide resistance, high bacterial load, and co-infections with other pathogens⁶. Macrolide-resistant M. pneumoniae (MRMP) has been associated with prolonged duration of illness, disease severity, and treatment failure, and is a major therapeutic dilemma, especially in Asia^7,8.

The prevalence of MRMP is a global concern, with Asia bearing the highest burden. It was first reported in Japan, and since then, its incidence has been on the rise globally^9,10. The prevalence of MRMP in Asia, North America, South America, Europe, and Oceania is 63%, 8.6%, 0%, 3% and 3.3%, respectively¹¹. This trend is partly attributed to the long-term course of macrolide therapy and widespread dissemination of resistant strains¹².

Multilocus sequence typing (MLST) has proven to be a valuable research tool for the study of bacterial pathogens, enabling genetic population analysis, species definition, and correct identification of isolates. Brown et al. have created an MLST scheme for M. pneumoniae using eight highly discriminatory housekeeping genes¹³. Although MLST has been effectively applied to M. pneumoniae in various countries, no such studies have been conducted in Iran so far^14,15.

Due to the limited data on MRMP in our region, this study aimed to investigate antimicrobial susceptibility and molecular typing of M. pneumoniae isolates, obtained from outpatients with atypical pneumonia.

Results

Prevalence of M. pneumoniae in patients with atypical pneumonia and the clinical features

Among 270 patients with atypical pneumonia, there were 104 males and 166 females with a median age of 49 years (IQR 32-63). Dry cough was the most prevalent symptom, observed in 85.2% of cases, followed by chest pain (66.7%), dyspnea (48.1%), headache (27.8%), sore throat (25.9%), sputum production (24.1%), and fever (11.1%). M. pneumoniae was detected in 20 of 104 males (19.2%) and 34 of 166 females (20.5%). No statistically significant association was observed between sex and the prevalence of M. pneumoniae infection (p = 0.79).

Antimicrobial susceptibility testing

The majority of isolates (n = 46; 85.1%) exhibited high MIC values for macrolides. The MIC₅₀ and MIC₉₀ values for azithromycin were 32 μg/ml and > 64 μg/ml, respectively, indicating reduced efficacy across the majority of isolates. Erythromycin exhibited an even higher MIC₅₀ of 64 μg/ml, also with an MIC₉₀ > 64 μg/ml. Three isolates (5.5%) were resistant to clindamycin, with MICs of 8 μg/ml, and five isolates (9.25%) showed reduced susceptibility to tetracycline, with MICs of 4 μg/ml. In contrast, all isolates remained susceptible to levofloxacin, with MIC values ≤ 0.25 μg/ml, highlighting it as the most effective agent in this study. Detailed data on antimicrobial susceptibility and MIC breakpoint values are presented in Table 1.

Table 1.

Antimicrobial activity of five antimicrobial agents against 54 clinical Mycoplasma pneumoniae strains.

Antimicrobial agent	Breakpoints (μg/ml)			MIC50 (µg/mL)	MIC90 (µg/mL)	Non-susceptible Isolates (%)	Isolates with reduced susceptibility (%)
	S	I	R	R	R
Erythromycin	≤ 0.5	–	≥ 1	64	≥ 64	85.1	–
Azithromycin	≤ 0.5	–	≥ 1	32	≥ 64	85.1	–
Clindamycin	≤ 0.25	–	≥ 0.5	0.125	0.25	5.5	–
Levofloxacin	≤ 1	–	–	0.125	0.5	–	0
Tetracycline	≤ 2	–	–	0. 25	0.5	–	9.25

S: susceptible; I: intermediate; R: resistant.

MIC50: minimum inhibitory concentration required to inhibit the growth of 50% of isolates.

MIC90: minimum inhibitory concentration required to inhibit the growth of 90% of isolates.

The associations between demographic characteristics, clinical features of patients, and antibiotic resistance of M. pneumoniae were evaluated. Only a limited number of clinical variables demonstrated statistically significant relationships with antibiotic resistance. Regarding macrolide resistance, headache was significantly less common among patients infected with resistant strains (p = 0.004, OR = 0.081, 95% CI 0.01–0.40). For tetracycline resistance, several clinical symptoms were positively associated with increased odds of resistance. Headache (p = 0.018, OR = 13.81, 95% CI 1.39–136.69), vomiting (p = 0.004, OR = 35.25, 95% CI 3.60–345.02), and sore throat (p = 0.013, OR = 15.60, 95% CI 1.56–155.41) were significantly more frequent among patients with resistant strains. Conversely, cough was less prevalent in resistant cases (p = 0.001, OR = 0.022, 95% CI 0.002–0.24). For clindamycin resistance, headache was the only symptom with a statistically significant association (p = 0.018, OR = 1.25, 95% CI 0.97–1.61), although the confidence interval suggests a borderline effect. Other features did not reach statistical significance. See Table 2.

Table 2.

Demographic data and clinical characteristics of patients with M. pneumoniae and their association with antibiotic resistance.

Characteristics		Macrolides				Tetracycline				Clindamycin
		Resistant strains (n = 46)	P-value	OR	95% CI	Resistant strains (n = 5)	P-value	OR	95% CI	Resistant strains (n = 3)	P-value	OR	95% CI
Gender	Male (n = 20)	18 (90%)	0.69	0.519	0.09–2.85	0 (0%)	0.14	1.172	1.02–1.34	0 (0%)	0.29	1.097	0.98–1.21
	Female (n = 34)	28 (82.4%)				5 (14.7%)				3 (8.8%)
Age	1–20 (n = 5)	5 (100)	> 0.90	0.837	0.73–0.94	1 (20%)	0.39	2.813	0.25–31.57	1 (20%)	0.26	5.875	0.43–79.77
	21–40 (n = 8)	8 (100%)	0.33	0.826	0.72–0.94	0 (0%)	> 0.9	0.891	0.80–0.98	0 (0%)	> 0.9	0.935	0.86–1
	41–60 (n = 18)	14 (77.8%)	0.41	0.438	0.09–2	3 (16.7%)	0.32	3.4	0.51–22.49	2 (11.1%)	0.25	4.375	0.36–51.84
	61–90 (n = 23)	19 (82.6%)	0.71	0.704	0.15–3.17	1 (4.3%)	038	0.307	0.77–2.94	0 (0%)	0.25	0.903	0.80–1.01
Clinical features of patients with resistant M. pneumoniae strains	Fever (n = 6)	6 (100%)	0.57	0.833	0.73–0.94	0 (0%)	> 0.9	0.896	0.81–0.98	0 (0%)	> 0.9	0.938	0.87–1
	Chest pain (n = 36)	30 (83.3%)	0.70	0.625	0.11–3.46	4 (11.1%)	0.65	2.125	0.22–20.54	2 (5.6%)	> 0.9	1	0.08–11.82
	Headache (n = 15)	9 (60%)	0.004	0.081	0.01–0.4	4 (26.7%)	0.02	13.81	1.39–136.69	3 (20%)	0.02	1.25	0.97–1.61
	Vomit (n = 5)	5 (100%)	> 0.9	0.837	0.73–0.94	3 (60%)	0.004	35.250	3.60–345.02	1 (20%)	0.26	5.875	0.43–79.77
	Sore throat (n = 14)	12 (85.7%)	> 0.9	1.059	0.18–5.97	4 (28.6%)	0.01	15.600	1.56–155.41	2 (14.3%)	0.16	6.5	0.54–78.09
	Dry cough (n = 46)	38 (82.6%)	0.33	1.211	1.06–1.38	1 (2.2%)	0.001	0.022	0.002–0.24	1 (2.2%)	0.05	0.067	0.005–0.85
	Sputum (n = 13)	13 (100%)	0.17	0.805	0.69–0.93	1 (7.7%)	> 0.9	0.771	0.07–7.58	1 (7.7%)	> 0.9	1.625	0.13–19.52
	Dyspnea (n = 26)	22 (84.6%)	> 0.9	0.917	0.20–4.11	3 (11.5%)	0.66	1.696	0.26–11.05	2 (7.7%)	0.60	2.25	0.19–26.40

OR: odds ratio; 95% CI 95% confidence interval; The bold values in the table are the indicators that showed a p-value < 0.05.

MLST analysis

MLST was performed on 20 MRMP isolates, revealing eight distinct STs. The most common type was ST3, detected in 10 isolates (50%), followed by ST14 (15%) and ST2 (10%) (Table 3). The association between STs and resistance to macrolide antibiotics was established using the chi-square test. The results revealed a statistically significant correlation between STs and MRMP isolates. Specifically, MRMP isolates were found to be 1.92 times more likely to exhibit ST3 compared to other STs (P = 0.001).

Table 3.

Distribution of clonal complexes, sequence type profiles of macrolide-resistant Mycoplasma Pneumoniae (MRMP) isolates from patients with atypical pneumonia in Tehran.

Isolate	Gender	CC	ST	Allelic profiles
				ppa	pgm	gyrB	gmk	glyA	atpA	arcC	adk
M-90	Male	3	14	2	3	2	2	4	4	1	5
M-82	Male	1	3	1	2	1	1	1	3	1	1
M-75	Female	1	3	1	2	1	1	1	3	1	1
M-71	Female	1	3	1	2	1	1	1	3	1	1
M-70	Male	1	3	1	2	1	1	1	3	1	1
M-60	Male	1	3	1	2	1	1	1	3	1	1
M-55	Female	3	14	2	3	2	2	4	4	1	5
M-43	Female	1	20	1	2	1	3	1	3	1	1
M-33	Female	1	3	1	2	1	1	1	3	1	1
M-30	Male	1	3	1	2	1	1	1	3	1	1
M-27	Female	1	3	1	2	1	1	1	3	1	1
M-26	Female	2	31	2	8	2	2	2	4	1	1
M-22	Female	1	17	1	5	1	1	1	3	1	1
M-18	Male	2	2	2	3	2	2	2	4	1	1
M-157	Female	1	19	1	6	1	1	1	3	1	1
M-140	Male	3	14	2	3	2	2	4	4	1	5
M-134	Male	2	2	2	3	2	2	2	4	1	1
M-126	Male	1	3	1	2	1	1	1	3	1	1
M-106	Female	1	3	1	2	1	1	1	3	1	1
M-102	Female	3	32	2	3	2	2	7	4	1	5

CC: clonal complex; ST: sequence type.

To visualize the genetic relatedness among the STs, a minimum spanning tree was generated with PhyloViz and presented in Fig. 1A. Clonal complex (CC) assignment using the goeBURST algorithm identified three distinct clonal complexes. Specifically, 13 isolates (65%) were related to clonal complex-1 (CC1), while three isolates (15%) belonged to clonal complex-2 (CC2), and four isolates (20%) were classified in the third clonal complex (CC3) (Fig. 1B and Supplementary Data 1).

Fig. 1.

Minimum spanning tree and clonal complex assignment of macrolide-resistant Mycoplasma pneumoniae isolates. (A) MST constructed based on MLST allelic profiles of 20 MRMP isolates using BioNumerics software. Each circle represents a distinct ST, with the size proportional to the number of isolates. Lines indicate the number of allelic differences between STs. (B) Clonal complex (CC) analysis performed using the goeBURST algorithm. STs were grouped into three main clonal complexes: CC1 (13 isolates), CC2 (3 isolates), and CC3 (4 isolates), based on single locus variants (SLV) and allelic relationships.

Phylogenetic analysis

A phylogenetic tree was constructed based on the concatenated sequences of the eight MLST housekeeping genes using the Unweighted Pair Group Method with Arithmetic Mean (UPGMA) algorithm in BioNumerics. As shown in Fig. 2, the tree clearly divides the isolates into three major clusters, which correspond to the clonal complexes identified in the goeBURST analysis (CC1, CC2, and CC3). The bootstrap values for the major branches were all above 90%, indicating high reliability of the clustering structure. No outlier or singleton STs were found. The structure of the tree closely resembles the goeBURST results, further validating the clonal relationships inferred from allelic profiles.

Fig. 2.

UPGMA phylogenetic tree based on concatenated MLST sequences of M. pneumoniae isolates. The tree shows three main clusters corresponding to clonal complexes CC1, CC2, and CC3, consistent with the goeBURST analysis. No singleton or outlier STs were detected.

Discussion

The rising prevalence of MRMP poses a major therapeutic challenge, particularly in Asian countries where the burden of infection is notably higher¹⁶. Our study provides updated and region-specific evidence from Iran, addressing an important gap in the epidemiological data on M. pneumoniae. We found an alarmingly high prevalence of macrolide resistance among circulating isolates in Tehran, while all strains remained susceptible to levofloxacin. Furthermore, MLST analysis revealed limited yet notable genetic diversity, with ST3 as the dominant sequence type and CC1 identified as the major clonal complex. These findings highlight both the clinical importance of antimicrobial resistance in M. pneumoniae and the need for continued surveillance of its evolving molecular epidemiology.

Our study identified identical resistance rates (85.1%) for erythromycin and azithromycin, likely due to their structural similarities and overlapping mechanisms of action, which facilitate cross-resistance among macrolide antibiotics. The two drugs interact with the bacterial ribosome’s 23S rRNA subunit, and A2058G and A2059G gene mutations have beenconsidered to render bacteria resistant to these drugs¹⁷. This high rate of resistance confirms that MRMP is a major clinical concern in Iran. Although this prevalence is higher than the previous national report with an MRMP prevalence rate of 56.9%¹⁸, it is comparable to rates reported in some East Asian countries. Recent studies in China¹⁹, Korea²⁰, and Japan⁹ have reported MRMP rates of 99.1%, 76.6%, and 54.1%, respectively. While USA²¹, Italy²², and Germany²³, it was 2.4%, 7.5%, and 3%, respectively. The elevated resistance rates in Asian countries are likely driven by the widespread empirical use of macrolides for respiratory infections, which exerts continuous selective pressure on circulating M. pneumoniae strains²⁴.

In contrast, all isolates remained fully susceptible to levofloxacin, supporting its potential as an alternative treatment option, especially in settings with high macrolide resistance. Nonetheless, its clinical use must be carefully considered due to age-related safety concerns¹⁰. The absence of fluoroquinolone resistance in our isolates aligns with global observations indicating that M. pneumoniae has not yet developed resistance to this class of antibiotics. However, genital Mycoplasma species have shown high resistance rates to fluoroquinolones²⁵.

Overall, these findings highlight a concerning therapeutic challenge in managing M. pneumoniae infections and underscore the necessity of antimicrobial stewardship strategies to mitigate further resistance development. Regarding clinical manifestations, our findings showed that most symptoms were not meaningfully associated with antibiotic resistance. This reinforces the need for laboratory testing as the primary and reliable method for accurately identifying resistant strains.

Moreover, epidemiological surveillance remains essential to control the infectious diseases. Among these, typing tools particularly sequencing-based methods have demonstrated high reliability, reproducibility, and ease of use^1,15. In this context, our study contributes valuable regional data by presenting the first phylogenetic analysis of M. pneumoniae isolates in Iran using a standardized MLST scheme.

Our study offers key insights into the genetic diversity, clonal distribution, and resistance patterns of M. pneumoniae in Iran. Surprisingly, ST3 was the most prevalent among macrolide-resistant isolates, accounting for 50% of MRMP cases. This finding aligns with previous reports from East Asia, including studies from South Korea and Japan, where ST3 has consistently been identified as the dominant clone during MRMP outbreaks^26,27. Similarly, other recent studies from Taiwan and China have found ST3 to be the dominant genotype among macrolide-resistant strains^19,28.

The clonal expansion of ST3 might suggest a potential fitness advantage, such as enhanced transmission competency or immune evasion mechanisms²⁶, although direct experimental tests are needed to confirm this. Widespread use of macrolides in Iran for the treatment of respiratory infections likely exerts selective pressure, promoting the survival and dissemination of resistant clones such as ST3. Considering that, in our study the MLST was performed only on resistant isolates, the presence or absence of ST3 among susceptible strains remains unknown. However, similar studies in Asia have noted the overrepresentation of ST3 in MRMP isolates²⁷. Moreover, due to the cross-sectional nature of the study, it is unclear whether ST3’s prevalence reflects recent clonal expansion or long-term persistence. Further genomic studies are needed to clarify its evolutionary dynamics and resistance mechanisms.

Moreover, the MRMP isolates were assigned to three CCs, with the predominance of CC1. This clonal complex consists of ST3, followed by ST17, ST19, and ST20. ST17 have been also repeatedly reported to correlate with macrolide resistance^19,29,30.

To put our results into a global perspective, we conducted a comparative analysis with information in the PubMLST database that has an extensive collection of M. pneumoniae sequence types and their corresponding antimicrobial resistance profiles³¹. PubMLST has reported a total of 506 isolates of 30 STs of M. pneumoniae globally to date. The most prevalent STs include ST3 (n = 263, 51.98%), ST14 (n = 75, 14.82%), ST2 (n = 38, 7.51%), ST19 (n = 24, 4.74%), and ST17 (n = 16, 3.16%). Aside from the predominance of ST3 in our study, the prevalence of ST2 and ST14 among resistant isolates was higher than has been documented globally. Such results suggest potential regional variations in M. pneumoniae distribution and resistance profiles, which should be investigated further for their reasons.

This study emphasizes the high necessity for continuous surveillance of the genetic diversity and antimicrobial susceptibility patterns of M. pneumoniae in Iran and worldwide. Studies like ours contribute to more targeted public health interventions by clarifying the relationships between sequence types, clonal complexes, and resistance patterns. However, several limitations should be acknowledged. MLST was conducted only on macrolide-resistant isolates, preventing direct comparison with susceptible strains. Moreover, the number of isolates analyzed was relatively limited, which may restrict conclusions regarding clonal diversity. In addition, the cross-sectional design does not clarify whether the observed sequence types represent recent expansion or long-term circulation. Considering these limitations, future studies should incorporate larger and more geographically diverse isolate collections and include both resistant and susceptible strains. In addition, comprehensive approaches such as whole-genome sequencing and evaluation of clinical outcomes might help better characterize the evolutionary dynamics and clinical significance of circulating M. pneumoniae genotypes.

Conclusion

This study provides novel insights into antimicrobial resistance and molecular epidemiology of MRMP in Iran. It highlights the genetic diversity and high rate of macrolide resistance among M. pneumoniae strains in Iran, with ST3 identified as the predominant macrolide-resistant clone. The findings emphasize the urgent need for ongoing molecular surveillance and rational antibiotic use to limit the spread of resistant strains. Understanding the relationship between sequence types and resistance can guide more effective and personalized treatment strategies in the future.

Methods

Criteria for patient selection

This study received ethical approval from the Research Ethics Committee of Tehran University of Medical Sciences (approval ID: TUMS.SPH.REC.1397.176). All methods were performed in accordance with the relevant guidelines and regulations and in compliance with the Declaration of Helsinki. Written informed consent was obtained from all participants involved in the study, who were informed about the purpose of the research, with participation entirely voluntary.

Patients with atypical pneumonia were included, characterized by dry cough, dyspnea, and fever (> 38 °C). Radiographic evidence of pneumonia was also considered for inclusion. Patients were excluded from the study if they developed pneumonia 72 h after hospital admission or within seven days following discharge, or had received macrolide antibiotics within 48 h prior to enrollment³². To ensure independent sampling, patients with proven epidemiological links, such as family members or individuals from the same institutional setting, were excluded.

Sample collection and identification of M. pneumoniae

A total of 270 patients with suspected atypical pneumonia were included from four outpatient clinics in Tehran. A throat swab was collected from each patient, and the presence of M. pneumoniae in clinical samples was confirmed by culture and polymerase chain reaction (PCR). DNA was extracted using the FavorPrep Tissue Genomic DNA Extraction Mini Kit (Favorgen Biotech Corporation, Taiwan), and PCR was performed using primers targeting the p1 adhesion gene, as previously described³². M. pneumoniae isolates were stored in PPLO broth supplemented with 5% horse serum, and containing 20% sterile glycerol at − 70 °C for further use.

Antimicrobial susceptibility testing

Minimum inhibitory concentration of four antimicrobial agents including erythromycin, azithromycin, clindamycin, and levofloxacin against M. pneumoniae isolates was determined using broth microdilution method according to M43-A Clinical and Laboratory Standards Institute (CLSI) guidelines³³. A two-fold serial dilution of each antibiotic was prepared in wells 1–8 of each row of a 96-well plate. Wells 9, 10, 11, and 12 were considered for solvent, media, drug, and growth controls, respectively. Then, 175 µl of bacterial inoculum (10⁴–10⁵ CFU/ml) was added to wells 1–9, and 12. For wells 10 and 11, 175 µl of broth media was added. Plates were incubated at 37 °C and read daily for a color change in the growth control well. The MIC was determined as the lowest concentration that inhibited the color change.

MLST analysis

For molecular typing, 20 MRMP isolates were selected using a stratified random sampling technique to ensure representativeness with minimal bias and optimal utilization of resources. After extracting the genomic content of the selected 20 MRMP strains, eight housekeeping genes (ppa, pgm, gyrB, gmk, glyA, atpA, arcC, and adk) were amplified by PCR and sequenced using an ABI 3730xl DNA Analyzer (Life Technologies)¹³. The allelic profile and ST of each isolate was identified by aligning the sequence of housekeeping genes against the PubMLST database (https://pubmlst.org)³¹.

To visualize the relationship between STs, a Minimum Spanning Tree (MST) was generated using the geoBURST algorithm in PHYLOViZ 2.0³⁴. Clonal complexes were defined as STs that shared at least 5 out of 7 alleles. For phylogenetic analysis, the gene sequences were concatenated and aligned. Then, a dendrogram was constructed using the Unweighted Pair Group Method with Arithmetic Mean (UPGMA) method in MEGA X³⁵. The UPGMA was visualized with the Interactive Tree of Life (iTOL) server³⁶.

Statistical analysis

Data analysis was performed using SPSS software version 24.0 (IBM Corp., Armonk, NY, USA). Associations between demographic or clinical variables and antibiotic resistance were assessed using Pearson’s χ² test or Fisher’s exact test when appropriate. Odds ratios (ORs) and their corresponding 95% confidence intervals (CIs) were calculated to estimate the strength of associations. A p-value < 0.05 was considered statistically significant.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

MA: methodology, conducting experiments; data interpretation, writing original draft; YS: validation, and writing—review & editing; NNG: preparing figures and tables, validation, and writing—review & editing; MRP: conceptualization, funding acquisition, methodology, supervision, validation, writing—review & editing.

Funding

This study was supported by the Tehran University of Medical Sciences, Tehran, Iran (Grant No. 34054).

Data availability

The datasets used and/or analyzed during the current study will be available from the corresponding author upon request.

Declarations

Competing interests

The authors declare no competing interests.

Ethics approval and consent to participate:

This study received ethical approval from the Research Ethics Committee of the Tehran University of Medical Sciences (approval ID: TUMS.SPH.REC.1397.176). Informed consent was obtained from all participants involved in the study. Participants were informed about the purpose of the research, and their participation was entirely voluntary.