Effect of COVID-19 pandemic on incidence of mycobacterial diseases among suspected tuberculosis pulmonary patients in Tehran, Iran.

Background: Recent pandemic of coronavirus SARS-CoV-2 (COVID-19) caused limitations in the country's strategies to fight against mycobacterial infections. The aim of this study was to compare the suspected tuberculosis (TB) pulmonary patients before and during the COVID-19 pandemic (January 2018-December 2021) who were referred to the National Reference TB Laboratory (NRL TB), Tehran, Iran. The mycobacterial isolated strains were identified and compared with previous data. Methods: A total of 16,899 clinical samples collected from 7041 suspected pulmonary TB patients were received from 2018 to 2021. Primary isolation of Mycobacterium isolates was done on Löwenstein-Jensen medium. Then, the DNA was extracted from acid-fast bacillus culture-positive samples and identification was performed by IS6110, Hsp65, and 16S-23S rRNA genes using polymerase chain reaction (PCR), PCR-restriction fragment length polymorphism, and nested PCR methods. Results: A total of 11679 specimens (69.1%) from 4866 suspected TB patients were collected in 2018-2019 and 5220 specimens (30.8%; from 2175 patients) in 2020-2021. Out of 11679 specimens, 2046 samples that belong to 852 patients were infected with Mycobacterium tuberculosis, and the remaining were non-TB Mycobacterium (NTM) species (n = 244) isolated from 102 patients. The cultures for 12894 specimens were either negative (76.3%) or contaminated (845/16899; 5%). A comparison of the total number of patients who were referred for diagnosis and treatment (954/666 patients, P > 0.05) showed a 30.1% reduction during the COVID-19 pandemic. Although, with these low number of patients, the significant increases of NTM species (P < 0.05) among suspected TB pulmonary patients were observed. Besides, new species of NTM, for example, Mycobacterium peregrinum and Mycobacterium montefiorense, were detected. For the past 20 years, these two species were not reported from pulmonary patients in Iran. Conclusions: During the pandemic of COVID-19, the TB diagnosis network became irregular, as a consequence, many patients could not reach the treatment center, and this could increase the circulation of mycobacterial diseases (TB and NTM). The study shows the emergence of new opportunistic NTM species also.

Correlation of single-nucleotide polymorphism at interferon-gamma R1 (at Position - 56) in positive purified protein derivative health workers with COVID-19 infection.

Background: The aim of the present study was to investigate the susceptibility of purified protein derivative (PPD) plus health-care workers to SARS-CoV-2 (COVID-19). For this reason, single-nucleotide polymorphism (SNP) of interferon-gamma (IFN-γ) gene at position +2109 and IFN-γ receptor 1 (R1) at position -56 was assessed in PPD plus group before and after COVID-19 infection (2017-2018; 2020-2021). Methods: The selected study cases (n = 100) that were working in tuberculosis (TB) unite (5-10 years) with PPD positivity >15 mm (16-20 mm) were included in this investigation. Sampling was done twice, once before and after the COVID-19 pandemic. Group A contains 50 samples collected from the GenBank TB laboratory that belong to TB staff before the pandemic (2017-2018). The other sample (Group B; 2021) was collected from the same unite during the COVID-19 pandemic. The SNP in the IFN-γ gene (+2109; 670 bp) and IFN-γ R 1 (-56; 366 bp) was performed using a specific primer and the polymerase chain reaction products were digested using restriction enzyme Fau I and Bts I, respectively. Statistical analyses were used to obtain the frequency of alleles among all studied cases. The confidence intervals (CIs) and t-test were calculated using the SPSS and GraphPad Prism software. Results: In overall, the most frequent genotype in Group A was AA (41/50; 82%) and Group B was 76% (38/50) in position + 2109 (odds ratio [OR] = 0.69, 95% CI, 0.26-1.83, and P = 0.46). Although in position -56, the most frequent genotype in Group A was TT (35/50; 70%) which significantly was than Group B TT (15/50; 30%) (OR = 0.184, 95% CI, 0.78-0.43, and P = 0.00). The frequency of allele A was more in both groups at position + 2109 (OR = 0.815, 95% CI, 0.23-2.86, and P = 0.75), whereas the dominate allele at position -56 was T in Group A (OR = 1.37, 95% CI, 0.62-3.02, and P = 0.42). Conclusion: No significant differences were observed in + 2109 in genotype among Group A and B. The main differences were seen in IFN-γ R1 at position (-56) between Group A and B. Hence, the IFN-γ R1 may play important role in COVID-19 infection. However, more study is needed to clear the IFN-γ correlation to COVID-19 infection.

Association of IL-10 gene promoter polymorphisms with susceptibility to pseudoexfoliation syndrome, pseudoexfoliative and primary open-angle glaucoma.

BACKGROUND: The involvement of cytokines in pathogenesis of pseudoexfoliation syndrome and glaucoma has been demonstrated in several studies. The aim of the present study was to explore the association between three promoter polymorphisms -592C/A (rs1800872), - 819C/T (rs1800871) and -1082A/G (rs1800896) of interleukin 10 (IL-10) gene with susceptibility to pseudoexfoliation syndrome (PEX), pseudoexfoliative glaucoma (PEXG), and primary open-angle glaucoma (POAG). METHODS: In this study, 114 PEX, 118 PEXG, 114 POAG patients and 126 healthy individuals from Iranian population were participated. Detailed ophthalmic examinations by an ophthalmologist including slit-lamp bio-microscopic examination, dilated examination of the lens, gonioscopy, and funduscopy were carried out on patients and controls. Genomic DNA was extracted from the blood samples and ARMS-PCR was performed to detect promoter polymorphisms of IL-10. RESULTS: In all three SNPs studied, there was a significant difference in the genotype distribution between patients and control subjects. Results revealed that the AA genotype of IL-10 -592C/A SNP is associated with PEX. However, TT genotype of -819C/T and AA genotype of -1082A/G SNP are significantly associated with susceptibility to either PEX or PEXG and POAG disorders. Furthermore, the ACC haplotype containing the IL-10 -1082A allele was associated with PEX (P = 0.02, OR = 5.76, 95% CI = 5.17-24.49), PEXG (P = 0.006, OR = 7.54, 95% CI = 6.62-30.76) and POAG (P = 0.003, OR = 8.11, 95% CI = 7.13-33.15). CONCLUSIONS: Our results demonstrated that IL-10 gene promoter polymorphisms are associated with susceptibility to PEX, PEXG and POAG in Iranian population. Considering the fact that IL-10 polymorphisms are associated with various IL-10 expressions, further research is needed to explain its involvement in these disorders and the formation of extracellular fibrillar amyloid deposits in PEX and PEXG.

TNFα -857 C/T and TNFR2 +587 T/G polymorphisms are associated with cystic fibrosis in Iranian patients.

Identification of modifier genes influencing phenotype of cystic fibrosis (CF) patients has become a challenge in CF pathophysiology, prognostic estimations and development of new therapeutic strategies. The aim of this study was to explore the association between four genetic polymorphisms of three modifier genes with CF, by comparing their alleles, genotypes and haplotype frequencies in patients and controls. In this favor, two regulatory polymorphic loci in TNFα promoter (-857C/T, rs1799724 and -238A/G, rs361525) and two functional polymorphic loci in TNFR1 (+36A/G, rs767455) and TNFR2 (+587T/G, rs1061622) were genotyped in 70 patients and 79 controls, using PCR-RFLP. Clinical pulmonary data were also recorded from all studied patients. Results indicated that an association was observed between both T allele and CT/TT genotypes of TNFα (P = 0.0005, OR = 7.06, 95% CI = 3.71-13.45) with CF under dominant model of inheritance. GG genotype of TNFR2 +587 (P = 0.0005, OR = 4.92, 95%CI = 2.34-10.34) was significantly associated with CF using recessive model. Consistently, more severe pulmonary disorder was found for patients carrying either T dominant allele of TNFα -857 or GG genotype of TNFR2 +587 polymorphic sites. Despite an association of A-T and G-T haplotypes with CF, no significant association was found between these haplotypes and clinical parameters of CF. Overall, TNFα -857 T allele and GG genotype of TNFR2 +587 were more frequent in CF patients compared to healthy controls and hence, they showed an association with CF and severe pulmonary phenotype in Iranian patients.

Association of Interferon-γ Receptor-1 Gene Polymorphism with Nontuberculous Mycobacterial Lung Infection among Iranian Patients with Pulmonary Disease.

Nontuberculous mycobacteria (NTM) cause significant pulmonary infections in humans. Researchers have reported an association between interferon-gamma receptor-1 (IFN-γR1 or IFNGR1) deficiency and susceptibility to NTM, but the relevance of polymorphism within these genes is not yet clear. In this study, a single nucleotide polymorphism (SNP), T to C, at position-56 in NTM patients with pulmonary disease was investigated. Molecular identification of Mycobacterium isolates was performed with hsp65 genes using polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP). Then, the host genomic DNA from confirmed NTM patients (N = 80) and control subjects (N = 80) were screened for SNPs of IFNGR1 (T-56C) by PCR-RFLP. The results indicated that NTM patients had higher TC (26/80; 32.5%) or CC (46/80; 57.5%) genotypes in comparison with control groups (TC genotypes [22/80, 27.5%]; CC genotypes [6/80, 7.5%]) (P < 0.05). In this regard, all the patients infected with rapid-growing Mycobacterium (RGM, i.e., Mycobacterium chelonae and Mycobacterium fortuitum) had CC genotypes (100%). In contrary, only 50.7% (35/69) of infected patients with slow-growing Mycobacterium (i.e., Mycobacterium simiae, Mycobacterium kansasii, and Mycobacterium avium-intracellulare) had CC genotypes. Thus, patients with CC mutation in IFNGR1 at position-56 are more likely to develop RGM infection. In overall, there is a significant association between SNP of IFNGR1 at position-56 and susceptibility to NTM infection. Based on these data, we propose SNP of IFNGR1 at position-56 as a suitable "biomarker" for identifying populations at higher risk of infection.

Susceptibility to pulmonary tuberculosis: host genetic deficiency in tumor necrosis factor alpha (TNF-α) gene and tumor necrosis factor receptor 2 (TNFR2).

OBJECTIVE/BACKGROUND: The susceptibility to tuberculosis (TB) depends upon different factors, and the risk of developing diseases after infection with Mycobacterium tuberculosis ranges from 5% to 10%. This suggests that besides the mycobacterial itself, the host genetic factors may determine the differences in host susceptibility to TB. Among the important risk factors, cytokines and especially tumor necrosis factor alpha (TNF-α) genes, are thought to be responsible for regulating the protective immune responses. The TNF-α gene that encodes the cytokines TNF-α is located within the class III region of the major histocompatibility complex (MHC). The TNF-α gene and its receptors have significant suppressive effects on bacterial growth into macrophages. Tumor necrosis factor receptor 1 (TNFR1) is more responsible when apoptosis is needed but tumor necrosis factor receptor 2 (TNFR2) is involved in cell survival. TNF-α conducts its replicative effects on immune cells via the latter. Up to now, several polymorphisms within the promoter region of the TNF-α gene have been shown to be associated with susceptibility or resistance to TB in different ethnic groups. By contrast, the correlation of TNF-α gene with their receptors such as TNFR2 in susceptibility to TB has not been resolved yet. In this study, we aimed to analyze the single nucleotide polymorphisms (SNPs) in the TNF-α gene at the -238, -308, -857, and -863 positions, and compare the susceptibility to TB with polymorphisms at TNFR2 (T587G). METHODS: One hundred fifty-one tuberculosis patients (n=151) and 83 control subjects (n=83) were included in this study. Polymorphisms in the TNF promoter region, namely TNF (SNP), -238, -308, -857, and -863 were studied using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). For TNFR2 polymorphisms at 587 positions, the following primers were used to amplify a 242 base pair (bp) product: 5'-TTCTGGAGTTGGCTGCGTGT-3' and ACTCTCCTATCCTGCCTGCT-3'. PCR products of TNFR2 digested with 2U enzymes of NLA III. RESULTS: The current study found a strong correlation between two polymorphisms in different loci of TNF-α gene including 857 C/C (85; 56.2%) and TNF 238 A/A 127 (84.1%). However, there were no associations between polymorphisms of the TNF-α gene and its receptor, that is, TNFR2. CONCLUSION: Concerning our current study, screening assessments for TNF-α-857 and A238GSNPs in Iran would be important in order to make future decisions for preventive treatments before getting the disease among individuals who are at high risk considering their genotyping.

Identification of different subtypes of rapid growing Atypical Mycobacterium from water and soil sources: Using PCR-RFLP using hsp65 and rRNA 16s-23s genes.

OBJECTIVE/BACKGROUND: Nontuberculosis mycobacteria (NTM) are a diverse group of microorganisms that cause a variety of diseases in humans including skin, respiratory, and gastrointestinal tract infection. Generally, NTM are classified into two categories: rapid (<7days) and slow growing (>7days). In this study, we aimed to investigate NTM frequency and prevalence in environmental samples. Additionally, we tried to identify various subtypes of isolated rapid growing mycobacteria (RGM). METHODS: Through a prospective descriptive cross-sectional study, water and soil samples were gathered from four neighboring towns around Tehran, the capital of Iran, at different geographic directions. Every 100m2 of the studied areas gave one sample containing 6g of soil in 3-5cm depth deposited in 50mL sterile water as sampling media. After digestion and decontamination, DNA from culture-positive specimens (RGM) were extracted using phenol-chloroform methods. Then the molecular identification of species and subspecies were performed using 16s-23s rRNA and hsp65 gene. RESULTS: In total, 341 RGM were found, out of which 322 (94.4%) were identified and 20 (5.8%) could not be identified. The most frequent RGM was, Mycobacterium fortuitum (72; 22%), Mycobacterium senegalense (58; 17.7%), Mycobacterium parafortuitum (44; 13.4%) and Mycobacterium conceptionense type 1 (24; 7.2%), and Mycobacterium cheloni type 1 (20; 6.0%). As shown in Table 1, M. fortuitum had more subtypes (8), and the frequency of subtypes 1 (27.7%), 4 (16.6%), and 5 (13.8%) were higher. Among subtypes of M. senegalense, subtype 1 had a higher frequency (70.4%) in comparison to subtype 2 (29.5%). M. cheloni had just one subtype. CONCLUSION: Our results showed M. fortuitum as the most prominent strain isolated from environmental samples. The frequency was similar in different places, irrespective of climatic variations. Availability of various subtypes of M. fortuitum might indicate a large circulation of this RGM in soil and water of Iranian territory. This high prevalence of M. fortuitum might raise the risk infection, especially in children, immunocompromised patients, diabetics, and cancer cases.

The importance of single nucleotide polymorphisms in interferon gamma receptor-1 gene in pulmonary patients infected with rapid grower mycobacterium.

OBJECTIVE/BACKGROUND: Interferon gamma (IFN-γ) plays a key role in protective immune response against Mycobacterial infection. IFN-γ excretes its antimycobacterial effectors mechanisms by activation of macrophages and dendritic cells via interaction with its receptor complex, that is, a ligand-binding subunit [IFN-γ receptor (IFNGR)1] and an accessory subunit (IFNGR2) on the cell surface. It has been shown that individuals with complete or partial IFNGR1 receptor deficiency are highly susceptible to infection by nontuberculous mycobacteria (NTM), Mycobacterium tuberculosis, and some Salmonella species. In the present study, we aimed to study the IFNGR1 T-56C single nucleotide polymorphism (SNP) in pulmonary patients that were infected with rapid grower mycobacterium. METHODS: Sputum specimens from suspected nontuberculosis pulmonary patients (n=95) were digested and decontaminated using 4% NaOH method. Molecular identification of mycobacterium was then performed by hsp65 genes using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Finally, the host genomic DNA from confirmed patients with rapid-grower mycobacterium (n=20) and control subjects (n=20) were screened for SNPs of IFNGR1 (T-56C) by PCR-RFLP. RESULTS: Out of 95 NTM patients, 20 (21.0%) were infected with rapid grower mycobacterium (RGM). The frequency of Mycobacterium chelonae (n=12) was more than Mycobacterium fortuitum (n=8), but the differences were not statistically significant. Interestingly, 18 patients (90%) had CC genotypes, whereas the remaining two had TC genotypes. The frequency of CC genotypes in the control group was <10% (p<0.05). CONCLUSION: There is a significant association between SNP of IFNGR1 at -56 and susceptibility to rapid grower infection.

Comparison of single nucleotide polymorphisms [SNP] at TNF-α promoter region with TNF receptor 2 (TNFR2) in susceptibility to pulmonary tuberculosis; using PCR-RFLP technique.

Since apoptosis and survival of the immune cells are crucially important in prevention or predisposition of individual from/to infections, especially in intracellular ones, the current study was performed to assess the correlation of host genetic polymorphisms with susceptibility to TB. For this reason, we investigated the difference of the single nucleotide polymorphisms [SNPs] in tumor necrosis factors [TNF-α] genes at (-238, -308, -857 and -863 position) and tumor necrosis factors receptors two [TNFR2] at (T 587 G position) between patients [n=151] and control [n=83]. The genotyping was studied by using PCR-RFLP which had high sensitivity in detecting compared with other techniques. The results showed a strong correlation between two polymorphisms in different loci of TNF-α gene including TNF-α T-857 C and A 238 G. But no association were found in TNFR2 genes with susceptibility to TB. And we found no correlation between TNFR2 and TNF-α gene polymorphisms. Therefore, the TNF-α T 857 C and A 238 G SNPs could be promising marker for identifying risk populations.