Interferon-gamma and interleukin-10 genetic polymorphisms among the pulmonary tuberculosis caused by Malaysia-specific Mycobacterium tuberculosis strain: SIT745/EAI1-MYS

Subjects

This study involved three groups of subjects: TB patients infected with M. tuberculosis strain SIT745/EAI1-MYS, TB patients infected with M. tuberculosis from other lineages (non-SIT745/EAI1-MYS), and healthy controls. Patients in the SIT745/EAI1-MYS group were specifically selected through initial screening of M. tuberculosis isolates. Archived M. tuberculosis isolates from the years 2011 to 2022 were screened using PCR-based typing to identify unique spacers defining the isolates as SIT745/EAI1-MYS (Ismail et al., 2014). Patients belonging to SIT745/EAI1-MYS isolates were traced and identified using the Laboratory Information System (LIS) of Hospital Universiti Sains Malaysia (USM). Similar procedures were carried out for subjects in the non-SIT745/EAI1-MYS group. Meanwhile, the healthy control group consisted of individuals with no history of tuberculosis. All subjects in this study were of Kelantanese Malay ethnicity and had no history of HIV positivity. Following the acquisition of written consent, demographic and clinical data were collected and recorded. A buccal swab was collected from each subject by rubbing a sterile cotton swab inside the cheeks for 20 s. Three buccal swab samples were collected from each subject and immediately suspended in a microcentrifuge tube containing 1 ml of 0.9% normal saline, then stored at 4 °C until used for DNA extraction. Research was conducted in the Research Laboratory, Department of Medical Microbiology and Parasitology, USM. This study was approved by the Human Research Ethics Committee, USM (Reference number: USM/JEPeM/18080362).

DNA extraction from buccal swab

DNA extraction from buccal swab samples was carried out using the QiaAmp DNA Mini kit (Qiagen, Hilden, Germany). Initially, the normal saline solution containing buccal mucosa cells was mixed by vortexing for 15 s and 400 μL of the cell suspension was added to AL lysis buffer in 1:1 ratio. The mixture was added with 20 μL of proteinase K solution and incubated at 56 °C for 15 min. After mixing with 400 μL of absolute ethanol, the whole mixture was transferred gradually to a spin column, underwent centrifugation and proceed with the remainder washing steps in accordance to the manufacturer’s guidelines. DNA was eluted using 50 μL of AE buffer and stored at −20 °C for molecular genotyping analysis.

Molecular genotyping by allelic-specific PCR

Molecular genotyping using allelic-specific PCR was performed to study the characteristics of genetic polymorphisms in IFN-γ (+874 A/T) and IL-10 (−1,082 A/G) genes separately.

To identify the polymorphism at +874 A/T of IFN-γ, two distinct reactions were established for each target. One tube contained a primer specific for the ‘A’ allele, while the other tube contained a primer specific for the ‘T’ allele. The presence or absence of bands at 261 bp for IFN-γ in these two tubes was noted. A parallel procedure was implemented for IL-10 (−1,082 A/G) allelic PCR, targeting a size of 550 bp. An internal control of size 426 bp was incorporated to verify the successful functionality of the PCR. The primers used were listed in Table 1.

A total volume of 25 μL PCR reaction mixture contained 5 μL of MyTaq Red reaction buffer (1×) and 0.3 µL of MyTaq Red DNA polymerase (0.5 units) (Bioline, Taunton, MA, USA), 1 μL of each sense and antisense primer (1 µM), 4.7 µL of dH₂O, and 3 µL of DNA template. The PCR reaction consisted of initial denaturation at 95 °C for 1 min, 10 cycles of denaturation at 95 °C for 15 s, annealing at 62 °C for 50 s, and elongation at 72 °C for 40 s, followed by 20 cycles 95 °C for 20 s, 56 °C for 50 s, and 72 °C for 50 s. An internal control of 426 bp was amplified using a pair of primers designed from the nucleotide sequence of the human growth hormone.

Statistical analysis

Data processing and analysis were conducted via statistical software SPSS version 24.0. The genotype polymorphisms between the control and TB patient groups were compared using the chi-squared test or Fisher’s exact test, as appropriate. When more than 20% of the expected counts are less than 5, the Fisher’s exact test was used instead of the Chi-squared test. Relative risk and odds ratio were calculated to assess disease susceptibility and clinical course. A p value < 0.05 was considered statistically significant. Due to the small sample size, post hoc effect size calculations for Cohen’s w and power analyses were also conducted based on the chi-squared test results to evaluate the strength of association and statistical robustness.

Demographic and clinical characteristics of the study participants

A total of 18 subjects, consisting of nine individuals from each group, consented to participate in this study. Table 2 shows the demographic data of TB patients and healthy control groups. Within the TB-SIT745/EAI1-MYS group, the majority were males (78%), aged less than 50 years (67%), and active smokers (44%). In contrast, the TB-non-SIT745/EAI1-MYS group primarily consisted of females (77.8%), aged over 50 years (67%), and non-smokers (78%). In this study, TB patients were included regardless of their diabetes mellitus (DM) status, due to the notably high prevalence among the Malay population in Kelantan, where the research was conducted. The inclusion of type 2 DM-positive individuals was deemed appropriate to ensure that the study reflects the real-world patient population in this region. Although DM may serve as a potential confounding factor in immunogenetic studies, the comparable proportion of DM-positive subjects in both TB-Malaysian strain and TB-non-Malaysian strain groups (67%) could reduce the risk of confounding bias for a more reliable analyses of genetic polymorphisms between these groups. On the other hand, hypertension was notably higher in the TB-non-SIT745/EAI1-MYS group (67%). The control group comprised healthy individuals with no tuberculosis history, with a slightly higher proportion of females (56%), individuals aged less than 50 years (56%), and the majority being non-smokers (89%).

Genetic polymorphisms in IFN-γ (+874 A/T) and IL-10 (−1,082 A/G) genes

Genetic polymorphisms in the IFN-γ (+874 A/T) and IL-10 (−1,082 A/G) genes were observed in all three groups, based on the presence or absence of the target amplicons (Fig. 1). For IFN-γ (+874 A/T), the presence of bands at 261 bp in both tubes indicated the AT genotype, while the presence of bands in only the first or second tube at 261 bp represented the AA genotype or the ‘TT’ genotype, respectively. The same pattern of observation was applied to the 550 bp target bands of IL-10 (−1,082 A/G). The results for IFN-γ (+874 A/T) and IL-10 (−1,082 A/G) genotype profiles were summarized in Table 3.

The correlation between IFN-γ +874 and IL-10 −1,082 genetic polymorphism and the odds of developing TB were analyzed as shown in Table 4. More than half (55.6%) of the TB patients infected with M. tuberculosis strains SIT745/EAI1-MYS were observed to carry +874 AA genotypes of IFN-γ, similar proportion to that of the healthy control group. The frequency of IFN-γ +874 A allele was higher than T allele in all three groups, including both TB patients and healthy controls. Among all, healthy controls carried a higher proportion of A allele, while patients infected with non-SIT745/EAI1-MYS strains carried the highest proportion of T allele. Interestingly, none of the healthy controls carried IFN-γ +874 TT homozygote genotype. This may indicate that individuals with IFN-γ +874 T allele might be more susceptible to tuberculosis infection. The distribution of genotypic and allelic frequencies of IFN-γ +874 A/T SNP in TB patients and healthy controls was not statistically significant (OR, 1.56; 95% CI [0.31–7.82]; p = 0.587) and (OR, 2.27; 95% CI [0.61–8.15]; p = 0.227).

Meanwhile, it was observed that the occurrence of the IL-10 (−1,082) AA genotype was 100% among healthy controls, 66.7% among SIT745/EAI1-MYS TB patients, and 77.8% among non-SIT745/EAI1-MYS TB patients. The heterozygous AG genotype was observed in 33.3% and 22.2% of the patients infected with SIT745/EAI1-MYS and non- SIT745/EAI1-MYS, respectively. None of the participants from all the three groups of this study carry the GG genotype. There are no significant differences in genotypic and allelic frequencies of IL-10 (−1,082 A/G) in all three groups (OR, 7.74; 95% CI [0.38–157.31]; p = 0.183) and (OR, 6.46; 95% CI [0.34–123.61]; p = 0.215).

To assess the statistical robustness of the observed associations despite the small sample size, post hoc effect size calculations and power analyses were performed. The calculated Cohen’s w for IFN-γ (+874 A/T) and IL-10 (−1,082 A/G) polymorphisms were 0.53 and 0.81, respectively, which indicate large effect sizes based on Cohen’s criteria (Ben Shachar et al., 2023). The large effect sizes suggest that the observed genetic variations may have meaningful associations with TB susceptibility in a broader population. Additionally, post hoc power analysis (1 – β) revealed moderate to high statistical power for the observed differences, with the power values of 0.65 for IFN-γ (+874 A/T) and 0.88 for IL-10 (−1,082 A/G). These findings support the relevance of the observed genotypes distributions despite the limited sample size, and underscore the importance of further studies with larger cohorts to validate these preliminary associations.

Analysis of TB severity between patients infected with SIT745/EAI1-MYS and non-SIT745/EAI1-MYS M. tuberculosis strains

The severity of TB was initially compared between patients infected with SIT745/EAI1-MYS and non-SIT745/EAI1-MYS M. tuberculosis strains based on the signs and symptoms as shown in Table 5. Overall findings demonstrated more or less similar degree of severity in both groups. Prolonged cough of more than two months was common for all the patients in both groups. However, a higher proportion of patients infected with SIT745/EAI1-MYS were observed to experience night sweats (77.8%) and loss of weight (77.8%) in comparison to non- SIT745/EAI1-MYS strains. Based on the chest radiograph reports, two patients from M. tuberculosis SIT745/EAI1-MYS infection had bilateral consolidation, whereas, other TB patients from both groups were observed to have unilateral consolidation. For TB patients infected with non-SIT745/EAI1-MYS strains, the symptoms for chest pain, hemoptysis, and shortness of breath were higher than those in the other group.

The scale for TB severity was assigned into mild, moderate, and severe, based on the types and the number of symptoms presence in each patient. Patients presented with less than four symptoms, excluding the hemoptysis and bilateral chest consolidation, were grouped into mild TB. Those with more than three symptoms and in the presence of shortness of breath were classified as moderate. Severe TB group comprised any patients presented with hemoptysis and bilateral parenchymal consolidation. Patients infected with SIT745/EAI1-MYS strain were recorded to have higher rate for severe TB (33.3%) compared to the non- SIT745/EAI1-MYS strains (22.2%).

Association of IFN-γ (+874 A/T) SNPs with demographic and clinical parameters in healthy controls and tuberculosis patients infected with SIT745/EAI1-MYS and non-SIT745/EAI1-MYS M. tuberculosis strains

The association of IFN-γ (+874 A/T) SNPs with demographic and clinical parameters were analyzed (Table 6). Overall, none of the healthy control and young participants of aged less than 50 years from both TB groups carry TT genotype. More than 77% of the TB patients were recorded with recent TB infections between the year 2017 and 2021. More than 60% of the patients who presented with shortness of breath were found to carry AT genotype.

More than half of the TB patients in the SIT745/EAI1-MYS group were IFN-γ (+874) AA genotype carriers (n = 5/9, 56%). Among the male patients in this groups, 57% (n = 4/7) carry the AA genotype, all of whom had underlying diabetes mellitus (DM). All the five AA genotype carriers in this group experienced weight loss, a majority of them (n = 4/5, 80%) had a low AFB smear index, which indicated a low amount of tuberculosis bacilli in their sputum specimens, and 80% (n = 4/5) presented with unilateral consolidation chest radiographs. None of them were recorded with shortness of breath. However, two patients with the AA genotype were classified as having severe TB due to hemoptysis and bilateral consolidation, respectively. Apart from the AA carriers, the SIT745/EAI1-MYS group included two individuals with IFN-γ (+874) AT genotype (22%) and two with TT genotype (22%). Both AT carriers had no underlying comorbidities and were non-active smokers, but they presented with shortness of breath, fever, loss of weight, and night sweats. One of these AT carriers had severe TB infections with bilateral consolidation in the chest radiograph (n = 1/2, 50%). The two patients with TT genotype in this group were both males aged over 50, active smokers, and had underlying DM. Both of the patients did not exhibit severe TB signs and symptoms.

On the contrary, almost half of the patients in the non-SIT745/EAI1-MYS group were IFN-γ (+874) AT carriers (n = 4/9, 45%), and all of them were female, non-smokers, and had recently acquired TB infections. These patients did not exhibit night sweats or bilateral consolidation laterality, and none of them had a third-degree AFB smear index. However, one of the patients was documented with hemoptysis and classified as having a severe TB infection (n = 1/4, 25%). Meanwhile, all three AA genotype carriers in this group were young TB patients (under 50 years old), and none of them had hemoptysis or bilateral consolidation chest findings. As for the two patients with the TT genotype in this group, both were over 50 years old and had underlying DM and HPT. Notably, one of these TT genotype carriers experienced hemoptysis and was recorded as having severe TB (n = 1/2, 50%).

Association of IL-10 (−1,082 A/G) SNPs with demographic and clinical parameters in healthy controls and tuberculosis patients infected with SIT745/EAI1-MYS and non-SIT745/EAI1-MYS M. tuberculosis strains

For the analyses of the association between IL-10 (−1,082 A/G) SNPs and the characteristics of healthy controls and TB patients, the findings in Table 7 showed that a majority of the participants possessed the IL-10 (−1,082) AA genotype (n = 22/27, 82%), which included all the healthy controls (n = 9/9, 100%) and 13 out of 18 TB patients. Whereas, none of the participants carried IL-10 (−1,082) GG genotypes.

Among the six patients with the IL-10 (−1,082) AA genotype in the SIT745/EAI1-MYS group, all were male with a history of smoking. A majority of them (n = 5/6, 83%) were over 50 years old and had underlying DM. None of the AA genotype carriers exhibit bilateral consolidation, but one of them experienced hemoptysis and was classified as having severe TB (n = 1/6, 17%). Meanwhile, the three IL-10 (−1,082) AG genotype carriers in this group comprised both females and one male patient. Two of them had severe TB (n = 2/3, 67%) due to bilateral consolidation chest findings.

In the non-SIT745/EAI1-MYS group, the seven IL-10 (−1,082) AA genotype carriers were predominantly females (n = 6/7, 86%), non-smokers (n = 6/7, 86%), and had underlying DM and HPT (n = 5/7, 71%). In terms of signs and symptoms, 57% (n = 4/7) developed shortness of breath, but none of them presented with bilateral chest radiographs. One patient with the IL-10 (−1,082) AA had severe TB with hemoptysis (n = 1/7, 14%). The IL-10 (−1,082) AG carriers in this group comprised one male (n = 1/2, 50%) and one female (n = 1/2, 50%) patient. Besides gender, this group had a balanced distribution of age group, comorbidities, and smoking history. Both patients experienced weight loss and a third-degree AFB smear. One of them was recorded as having severe TB due to hemoptysis (n = 1/2, 50%).

Association of IFN-γ and IL-10 genetic polymorphisms with demographic and clinical parameters among TB patients

For this analysis, this study aimed to determine the prevalence of dominant AA alleles and the recessive AT/TT or AG alleles for the respective IFN-γ and IL-10 genetic polymorphisms and their association with various demographic and clinical parameters among the TB patients (Table 8).

For IFN-γ +874 SNPs, the dominant AA alleles were prevalent among the males (n = 5/8, 63%), young age group (n = 5/8, 63%), those with smoking history (n = 5/8, 63%) and underlying DM (n = 5/8, 63%). More than half of these IFN-γ (+874) homozygous A carriers (n = 5/8, 63%) were infected by the SIT745/EAI1-MYS M. tuberculosis strain. Regarding the TB symptoms, patients with this IFN-γ (+874) homozygous A alleles were less likely to develop shortness of breath (n = 7/8, 88%) and night sweats (n = 5/8, 63%). Most of them experienced weight lost (n = 7/8, 88%) and had unilateral chest findings (n = 7/8, 88%), while 25% (n = 2/8) of these AA allele carriers developed severe TB symptoms.

In contrast, 56% of the patients (n = 10/18), especially females, carried either homozygous or heterozygous T allele at the IFN-γ (+874) SNPs. The majority of these T allele carriers belonged to the elderly group (n = 8/10, 80%) and had comorbidities with DM and HPT. It was also found that 60% of the patients with IFN-γ (+874) T allele had acquired TB due to a non-SIT745/EAI1-MYS M. tuberculosis infection. The T allele carriers had also been the most prevalent in developing shortness of breath (n = 6/10, 60%), and 30% (n = 3/10) of them had severe TB infections characterized by hemoptysis and bilateral pulmonary infections.

As for the IL-10 (−1,082) A/G SNPs, 72% of the patients (n = 13/18) carried the homozygous AA allele. This majority comprised predominantly elderly individuals suffering from both DM and hypertension (HPT). There is no significant association between the IL-10 (−1,082) AA carriers and the acquisition of specific M. tuberculosis strains. In terms of symptoms, all the IL-10 (−1,082) AA carriers had unilateral pulmonary infections, and a majority of them developed mild and moderate TB symptoms. Only 15% of the IL-10 (−1,082) AA carriers had severe TB with hemoptysis.

Of the total 18 patients, only five IL-10 (−1,082) heterozygous G carriers were found, and the majority were young females with no comorbidities (n = 3/5, 60%). Additionally, 60% of these carriers had acquired infections due to the SIT745/EAI1-MYS M. tuberculosis strain. Fever and weight loss were common among all the G allele carriers, with 60% (n = 3/5) of them developed severe TB with bilateral parenchymal consolidation and hemoptysis.