The importance of heteroresistance and efflux pumps in bedaquiline-resistant Mycobacterium tuberculosis isolates from Iran.

BACKGROUND: Tuberculosis (TB) continues to pose a threat to communities worldwide and remains a significant public health issue in several countries. We assessed the role of heteroresistance and efflux pumps in bedaquiline (BDQ)-resistant Mycobacterium tuberculosis isolates. METHODS: Nineteen clinical isolates were included in the study, of which fifteen isolates were classified as MDR or XDR, while four isolates were fully susceptible. To evaluate BDQ heteroresistance, the Microplate Alamar Blue Assay (MABA) method was employed. For screening mixed infections, MIRU-VNTR was performed on clinical isolates. Mutations in the atpE and Rv0678 genes were determined based on next-generation sequencing data. Additionally, real-time PCR was applied to assess the expression of efflux pump genes in the absence and presence of verapamil (VP). RESULTS: All 15 drug-resistant isolates displayed resistance to BDQ. Among the 19 total isolates, 21.05% (4/19) exhibited a heteroresistance pattern to BDQ. None of the isolates carried a mutation of the atpE and Rv0678 genes associated with BDQ resistance. Regarding the MIRU-VNTR analysis, most isolates (94.73%) showed the Beijing genotype. Fifteen (78.9%) isolates showed a significant reduction in BDQ MIC after VP treatment. The efflux pump genes of Rv0676c, Rv1258c, Rv1410c, Rv1634, Rv1819, Rv2459, Rv2846, and Rv3065 were overexpressed in the presence of BDQ. CONCLUSIONS: Our results clearly demonstrated the crucial role of heteroresistance and efflux pumps in BDQ resistance. Additionally, we established a direct link between the Rv0676c gene and BDQ resistance. The inclusion of VP significantly reduced the MIC of BDQ in both drug-susceptible and drug-resistant clinical isolates.

miR-22 gene therapy treats HCC by promoting anti-tumor immunity and enhancing metabolism.

MicroRNA-22 (miR-22) can be induced by beneficial metabolites that have metabolic and immune effects, including retinoic acids, bile acids, vitamin D3, and short-chain fatty acids. The tumor suppressor effects of miR-22 have been suggested, but whether miR-22 treats orthotopic hepatocellular carcinoma (HCC) is not established. The role of miR-22 in regulating tumor immunity is also poorly understood. Our data showed that miR-22 delivered by adeno-associated virus serotype 8 effectively treated HCC. Compared with FDA-approved lenvatinib, miR-22 produced better survival outcomes without noticeable toxicity. miR-22 silenced hypoxia-inducible factor 1 (HIF1α) and enhanced retinoic acid signaling in both hepatocytes and T cells. Moreover, miR-22 treatment improved metabolism and reduced inflammation. In the liver, miR-22 reduced the abundance of IL17-producing T cells and inhibited IL17 signaling by reducing the occupancy of HIF1α in the Rorc and Il17a genes. Conversely, increasing IL17 signaling ameliorated the anti-HCC effect of miR-22. Additionally, miR-22 expanded cytotoxic T cells and reduced regulatory T cells (Treg). Moreover, depleting cytotoxic T cells also abolished the anti-HCC effects of miR-22. In patients, miR-22 high HCC had upregulated metabolic pathways and reduced IL17 pro-inflammatory signaling compared with miR-22 low HCC. Together, miR-22 gene therapy can be a novel option for HCC treatment.

Tuberculosis prevention, diagnosis, and treatment financial profile during 2006-2021: PART A.

BACKGROUND: Tuberculosis (TB) is a communicable disease that is a major cause of death and one of the leading causes of death worldwide. Currently, there is no analyzed data to examine the financial profile of TB by country, continent, and year; this article analyzed TB prevention, diagnosis, and treatment financial profile during the last two decades. METHODS: Original research, reviews, and governmental databases are analyzed to present the financial profile of TB. RESULTS: Analyzed data showed Europe (23137.133), Asia (20137.073), and Africa (15237.973) had the most allocated funds (US $ million), and Oceania (236.702), and America (4745.043) had the lowest allocated fund (US $ million) during 2006-2021. Additionally, the allocation of funds (domestic funds, global funds, and grants [excluding global funds]) in different countries and proper planning for TB eradication has caused that in the last two decades, the slope of the confirmed cases and deaths graph line is negative. CONCLUSION: The number of confirmed cases and deaths reported globally is decreasing. The trend lines showed that the assigned funds are increasing, indicating that the TB eradication plan can be apprehended soon.

Identification of a novel fully human anti-toxic shock syndrome toxin (TSST)-1 single-chain variable fragment antibody averting TSST-1-induced mitogenesis and cytokine secretion.

BACKGROUND: Staphylococcal superantigens are virulence factors that help the pathogen escape the immune system and develop an infection. Toxic shock syndrome toxin (TSST)-1 is one of the most studied superantigens whose role in toxic shock syndrome and some particular disorders have been demonstrated. Inhibiting TSST-1 production with antibiotics and targeting TSST-1 with monoclonal antibodies might be one of the best strategies to prevent TSST-1-induced cytokines storm followed by lethality. RESULTS: A novel single-chain variable fragment (scFv), MS473, against TSST-1 was identified by selecting an scFv phage library on the TSST-1 protein. The MS473 scFv showed high affinity and specificity for TSST-1. Moreover, MS473 could significantly prevent TSST-1-induced mitogenicity (the IC50 value: 1.5 µM) and cytokine production. CONCLUSION: Using traditional antibiotics with an anti-TSST-1 scFv as a safe and effective agent leads to deleting the infection source and preventing the detrimental effects of the toxin disseminated into the whole body.

BK polyomavirus in Iranian patients with papillary thyroid cancer: Is it a major future challenge?

BK polyomavirus (BKPyV) is a well-known cause of nephropathy in renal transplant recipients. It has recently received much attention from researchers as a major predisposing factor for various cancers. This study aimed to investigate how BKPyV affected the advancement of papillary thyroid carcinoma (PTC). A total of 1057 samples were tested for BKPyV DNA and RNA, comprising 645 paraffin-embedded PTC biopsy samples (PEBS), 412 fresh biopsy samples (FBS), and 1057 adjacent noncancerous samples. The BKPyV DNA was found in 511 (48.3%) of the specimens, including 347 (84.2%) FBS and 164 (25.4%) PEBS. The mean BKPyV copy number was significantly lower in patients with PEBS (0.5 × 10-4 ± 0.1 × 10-4 copies/cell) than in FBS (1.3 × 10-1 ± 0.2 × 10-1 copies/cell) and non-PTC normal samples (0.3 × 10-5 ± 0.04 × 10-5 copies/cell). The PEBS had lower LT-Ag RNA expression than FBS, and no VP1 gene transcript expression was detected. In conclusion, although our findings indicated the presence of BKPyV in some Iranian PTC patients, more research is needed to corroborate these findings.

Strain-specific behavior of Mycobacterium tuberculosis in A549 lung cancer cell line.

BACKGROUND: A growing body of evidence has shown the association between tuberculosis (TB) infection and lung cancer. However, the possible effect of strain-specific behavior of Mycobacterium tuberculosis (M.tb) population, the etiological agent of TB infection in this association has been neglected. In this context, this study was conducted to investigate this association with consideration of the genetic background of strains in the M.tb population. RESULTS: We employed the elastic net penalized logistic regression model, as a statistical-learning algorithm for gene selection, to evaluate this association in 129 genes involved in TLRs and NF-κB signaling pathways in response to two different M.tb sub-lineage strains (L3-CAS1and L 4.5). Of the 129 genes, 21 were found to be associated with the two studied M.tb sub-lineages. In addition, MAPK8IP3 gene was identified as a novel gene, which has not been reported in previous lung cancer studies and may have the potential to be recognized as a novel biomarker in lung cancer investigation. CONCLUSIONS: This preliminary study provides new insights into the mechanistic association between TB infection and lung cancer. Further mechanistic investigations of this association with a large number of M.tb strains, encompassing the other main M.tb lineages and using the whole transcriptome of the host cell are inevitable.

Extracellular vesicles and pasteurized cells derived from Akkermansia muciniphila protect against high-fat induced obesity in mice.

BACKGROUND: Several studies have shown that probiotics have beneficial effects on weight control and metabolic health. In addition to probiotics, recent studies have investigated the effects of paraprobiotics and postbiotics. Therefore, we evaluated the preventive effects of live and pasteurized Akkermansia muciniphila MucT (A. muciniphila) and its extracellular vesicles (EVs) on HFD-induced obesity. RESULTS: The results showed that body weight, metabolic tissues weight, food consumption, and plasma metabolic parameters were increased in the HFD group, whereas A. muciniphila preventive treatments inhibited these HFD. The effects of pasteurized A. muciniphila and its extracellular vesicles were more noticeable than its active form. The HFD led to an increase in the colonic, adipose tissue, and liver inflammations and increased the expression of genes involved in lipid metabolism and homeostasis. Nevertheless, these effects were inhibited in mice that were administered A. muciniphila and its EVs. The assessment of the gut microbiota revealed significant differences in the microbiota composition after feeding with HFD. However, all treatments restored the alterations in some bacterial genera and closely resemble the control group. Also, the correlation analysis indicated that some gut microbiota might be associated with obesity-related indices. CONCLUSIONS: Pasteurized A. muciniphila and its EVs, as paraprobiotic and postbiotic agents, were found to play a key role in the regulation of metabolic functions to prevent obesity, probably by affecting the gut-adipose-liver axis.

Variation in Mycobacterium tuberculosis population structure in Iran: a systemic review and meta-analysis.

BACKGROUND: Acquiring comprehensive insight into the dynamics of Mycobacterium tuberculosis (Mtb) population structure is an essential step to adopt effective tuberculosis (TB) control strategies and improve therapeutic methods and vaccines. Accordingly, we performed this systematic review and meta-analysis to determine the overall prevalence of Mtb genotypes/ sublineages in Iran. METHODS: We carried out a comprehensive literature search using the international databases of MEDLINE and Scopus as well as Iranian databases. Articles published until April 2020 were selected based on the PRISMA flow diagram. The overall prevalence of the Mtb genotypes/sublineage in Iran was determined using the random effects or fixed effect model. The metafor R package and MedCalc software were employed for performing this meta-analysis. RESULTS: We identified 34 studies for inclusion in this study, containing 8329 clinical samples. Based on the pooled prevalence of the Mtb genotypes, NEW1 (21.94, 95% CI: 16.41-28.05%), CAS (19.21, 95% CI: 14.95-23.86%), EAI (12.95, 95% CI: 7.58-19.47%), and T (12.16, 95% CI: 9.18-15.50%) were characterized as the dominant circulating genotypes in Iran. West African (L 5/6), Cameroon, TUR and H37Rv were identified as genotypes with the lowest prevalence in Iran (< 2%). The highest pooled prevalence rates of multidrug-resistant strains were related to Beijing (2.52, 95% CI) and CAS (1.21, 95% CI). CONCLUSIONS: This systematic review showed that Mtb populations are genetically diverse in Iran, and further studies are needed to gain a better insight into the national diversity of Mtb populations and their drug resistance pattern.

Pulmonary Infection Related to Mimivirus in Patient with Primary Ciliary Dyskinesia.

Primary ciliary dyskinesia is a rare autosomal recessive disorder that causes oto-sino-pulmonary disease. We report a case of pulmonary infection related to mimivirus in a 10-year-old boy with primary ciliary dyskinesia that was identified using molecular techniques. Our findings indicate that the lineage C of mimivirus may cause pneumonia in humans.

A case report of wrist synovial infection due to Mycobacterium jacuzzii, Iran.

BACKGROUND: Mycobacterium jacuzzii (M. jacuzzii) was first isolated in 2003 by insertion of breast implants in Tel Aviv, Israel. In this case report, we describe our experience in detection of M. jacuzzii using phenotypic and genotypic test of wrist synovial sample. CASE PRESENTATION: A 73-year-old woman complained of pain and swelling in the right wrist for 4 months. Her body temperature was 37-38 °C, and symptoms, such as pain, swelling, and some movement limitation, were reported. Clinical laboratory parameters showed an elevated C-reactive protein (CRP) level, erythrocyte sedimentation rate (ESR), and white blood cells (WBC) count. The sequences of hsp65, rpoB, 16S rDNA, and sodA genes indicated very high homology to M. jacuzzii. CONCLUSION: We report a case of synovial infection caused by M. jacuzzii in a patient with severe wrist pain in Iran, who was treated with amikacin, levofloxacin, and ethambutol. The outcomes of treatment after 8 months were positive, and no recurrence of infection was reported in the patient.

Application of MIRU-VNTR on smear slides: a shortcut for detection of polyclonal infections in tuberculosis patients.

Mixed (polyclonal) infections are one of the main problems in tuberculosis (TB) management. The best available method for detecting polyclonal infections in TB is mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR). According to multiple studies, MIRU-VNTR method can be applied to detect TB-related polyclonal infections in sputum samples or cultures. Setup of MIRU-VNTR on smear slides can be an efficient approach, regardless of the limitations of cultures and sputum samples in many laboratories. The present study aimed at investigating the diagnostic potential of MIRU-VNTR on smear slides in detecting mixed infections. Ziehl-Neelsen-stained microscopic slides were prepared from 14 clinical specimens. For amplifying 24 MIRU-VNTR loci, PCR assay was performed on the smear slides, clinical specimens, and cultures. Based on the 24-locus MIRU-VNTR analysis, polyclonal infections were reported in 42.85% of smear slides, while the corresponding rate was estimated at 57.1% (8/14) in the clinical samples. In the corresponding cultures, the rate of mixed infection was 7.14% (1/14). Use of smear slides can be a safe option for transferring clinical specimens between environmental and reference laboratories. Considering their significant impact on TB treatment, it is essential to diagnose mixed infections in low-resource countries with a high prevalence of mixed infections. The present findings show that direct MIRU-VNTR on smear slides can be conveniently used for the detection of mixed infections.

Exosomes in tuberculosis: Still terra incognita?

Today, diagnosis, vaccination, and treatment of tuberculosis (TB) remain major clinical challenges. Therefore, an introduction of new diagnostic measures and biomarkers is necessary to improve infection control. The ideal biomarker for TB infection can be defined as a host or pathogen-derived biomolecule, which is potent for identifying infection and determining its clinical stage. Exosomes, defined as cell-derived nanovesicles released into biological fluids, are involved in cell-cell communication and immune modulation. These vesicles have emerged as a new platform for improving the clinical diagnosis and prognosis of different infectious diseases and cancers. The role of these nanovehicles, as alternative biomarkers for the improvement of TB diagnosis and treatment, has been demonstrated in a significant body of literature. In this review, we summarized recent progress in the clinical application of exosome-based biomarkers in TB infection.

Comparative study of interruption of signaling pathways in lung epithelial cell by two different Mycobacterium tuberculosis lineages.

Alveolar epithelial cell (AEC) provides a replication niche for Mycobacterium tuberculosis. Based on the role of AEC in M. tuberculosis pathogenesis and existence of genetic diversity within this bacterium, we investigated interactions between AEC II and two different M. tuberculosis lineages. We have compared the transcriptome and cytokines/chemokines levels of A549 infected by M. tuberculosis lineage three and four using qRT-PCR and ELISA arrays, respectively. We showed different M.  tuberculosis strains induced changes in different effectors that involved in TLRs and NF-κB signaling pathways. We observed different reaction of the studied lineages specifically in pathogenesis, immune evasion mechanism, IL-12/IFN-γ axis, and autophagy. Similar behavior was detected in regarding to apoptosis, necroptosis, anti-inflammatory responses, and canonical inflammasome. Our findings contribute to elucidate more details in pathogenesis, immune evasion strategies, novel target and druggable pathway for therapeutic intervention, and host directed therapy in tuberculosis infection. Also, different M.  tuberculosis lineages-dependent host-pathogen interactions suggested using only one strain for this kind of research will be controversial.

Occult hepatitis C virus infection in patients with beta-thalassemia major: Is it a neglected and unexplained phenomenon?

Occult hepatitis C virus (HCV) infection (OCI) is described as the presence of viral genome in both hepatocytes and peripheral blood mononuclear cells (PBMCs) despite constant negative results on serum HCV RNA tests. Beta-thalassemia major (BTM) describes a group of inherited blood diseases. Patients with BTM require repeated blood transfusions, increasing the risk of exposure to infectious agents. We aimed to assess the prevalence of OCI in Iranian BTM patients and to identify the role of host factors in OCI positivity. A total of 181 BTM patients with HCV negative markers were selected. HCV RNA was tested in PBMCs using nested polymerase chain reaction assay. The positive samples were then genotyped via restriction fragment-length polymorphism (RFLP) and 5'-untranslated region sequencing. Six (3.3%) out of 181 BTM patients had viral HCV genomes in PBMC samples. Three (50.0%), two (33.3%), and one (16.7%) out of these six patients were infected with HCV-1b, HCV-1a, and HCV-3a, respectively. OCI positivity was significantly associated with the serum level of uric acid (P = 0.045) and ABO blood group (P = 0.032). Also, OCI patients had unfavorable IFNL3 rs12979860 TT, IFNL3 rs8099917 GG, IFNL3 rs12980275 GG, and IFNL4 ss469415590 ∆G/∆G genotypes. In conclusion, we indicated the low frequency of OCI in BTM patients. Nevertheless, more attention is warranted considering the importance of this infection. Also, further studies are necessary to determine the actual prevalence of OCI among BTM patients in Iran.

Pulmonary Infection Associated with Mycobacterium canariasense in Suspected Tuberculosis Patient, Iran.

Mycobacterium canariasense had only been isolated in humans from blood and contaminated catheters. We report a case of pulmonary disease associated with M. canariasense infection that was identified by multilocus sequence analysis; the illness was initially ascribed to M. tuberculosis. M. canariasense should be considered a cause of respiratory infection.

High Prevalence of Bedaquiline Resistance in Treatment-Naive Tuberculosis Patients and Verapamil Effectiveness.

In the regions where bedaquiline (BDQ) is introduced into the regimen, analysis of MIC and screening for preexisting resistance mutations could be crucial. The high prevalence of isolates with high BDQ MICs without prior exposure to BDQ was worrisome. It was also concluded that efflux pumps play a pivotal role in intrinsic BDQ resistance; therefore, the potential of verapamil as an adjunctive therapy to combat BDQ resistance should be investigated.

Genetic Diversity of Multi- and Extensively Drug-Resistant Mycobacterium tuberculosis Isolates in the Capital of Iran, Revealed by Whole-Genome Sequencing.

The emergence and spread of multidrug resistant (MDR) Mycobacterium tuberculosis complex (MTBC) strains is a critical global health problem. Between 2014 and 2018, 606 MTBC strains were isolated from 13,892 suspected pulmonary tuberculosis (TB) patients in Tehran, Iran, including 16 (2.6%) MDR-TB cases. A combination of phenotypic and genotypic methods (whole-genome sequencing) was employed for the identification of additional drug resistances and strain-to-strain genetic distances as a marker for recent transmission events. MDR and extensively drug-resistant (XDR) TB cases were almost exclusively infected by lineage 2/Beijing strains (14/16, P < 0.001). We further showed that recent transmission and/or recent introduction of lineage 2/Beijing strains contribute to high XDR-TB rates among all MDR-TB cases and should be considered an emerging threat for TB control in Tehran. In addition, the extensive pre-existing drug resistance profiles of MDR/XDR strains will further challenge TB diagnostics in the region.

Genetic diversity of Mycobacterium tuberculosis isolates causing pulmonary and extrapulmonary tuberculosis in the capital of Iran.

OBJECTIVES: Evaluation of the genetic diversity of Mycobacterium tuberculosis (M.tb) and determining if the association between a specific genotype and the site of infection is crucial. Accordingly, the current study aimed at comparing predominant M.tb genotypes in pulmonary (PTB) and extrapulmonary tuberculosis (EPTB) isolates circulating in the capital of Iran. METHODS: The genetic diversity of culture-confirmed PTB and EPTB isolates were evaluated by Spoligotyping and MIRU-VNTR (mycobacterial interspersed repetitive-unit-variable-number tandem-repeat) typing methods. Genotyping data were analyzed with SITVIT, MIRU-VNTRplus, and TBminer databases. To assess adjusted associations, chi-square/the Fisher exact test and multiple logistic regression model were applied. RESULTS: URAL2 (NEW-1) (28/88; 31.8%) and CAS1-DELHI (25/84; 29.8%) genotypes were predominant in EPTB and PTB strains, respectively. Based on MIRU-VNTR typing, 158 different MIRU-VNTR patterns were identified. Clustering rate and minimum estimate of the proportion of TB caused by recent transmission was 4.1% and 8.1%, respectively. CONCLUSIONS: The current study provided new insight into circulating genotypes of M.tb in PTB and EPTB patients in Tehran, Iran. This low percentage of TB transmission rate, demonstrated that mode of TB transmission was mainly associated with reactivation of latent TB rather than recently transmitted infection in this region. There was no significant difference in the association between the genotypes of M.tb strains and the site of the disease.

Evaluation of the expression of cytokines and chemokines in macrophages in response to rifampin-monoresistant Mycobacterium tuberculosis and H37Rv strain.

Macrophages are the primary phagocytes in the lungs and a part of the host defense system against Mycobacterium tuberculosis (Mtb), involved in the primary immune response. While several studies have assessed the effects of resistance to rifampin on Mtb physiology, the consequences of mutations in genes encoding the beta subunit of RNA polymerase (rpoB) for host-pathogen interactions remain poorly understood. In this study, rifampin-monoresistant (RMR) Mtb and H37Rv strains were used to infect the THP-1-derived macrophages. Real-time quantitative reverse transcription PCR assay was carried out to determine mRNA expression in 84 cytokine and chemokine genes. Production of specific cytokines and chemokines was measured by ELISA assay. In conclusion, the current study shed more light on the fitness cost of RMR strain and the potential effects of rpoB gene mutations on Mtb-host interactions. These results initially demonstrate that the Mtb carrying the rpoB-S450L can modulate macrophage responses to mediate bacterial survival.

Induction effects of Faecalibacterium prausnitzii and its extracellular vesicles on toll-like receptor signaling pathway gene expression and cytokine level in human intestinal epithelial cells.

A single layer of epithelial cells creates an interface between the host and microorganisms colonizing the gastrointestinal tract. In a healthy intestine, commensal bacteria and their metabolites can interact with epithelial cells as they are identified by Toll-like receptors (TLRs); This interaction results in homeostasis and immune responses. The present study aimed at evaluating Faecalibacterium prausnitzii- and extracellular vesicles (EVs)-induced expression of involved genes in TLRs signaling pathway and cytokines production in Caco-2 cell line. In this study, Caco-2 cell line was treated with F. prausitzii and its EVs. Using the protein levels of 12 cytokines were also evaluated by ELISA assay. F. prausnitzii induced upregulation in FOS, JUN, TNF-α, NFKB1, TLR3, IKBKB and CD86 genes. Furthermore, stimulation of Caco-2 cells with EVs derived from F. prausnitzii induced upregulation of CXCL8, CCL2, FOS, MAP2K4, TLR7, TLR3, IRF1, NFKBIA and TNF-α genes. Based on ELISA assay, Caco-2 cells treated with F. prausnitzii and its EVs showed a significant increase in TNF-α, IL-4, IL-8, and IL-10 expression and significant decreased in IL-1, IL-2, IL-6, IL-12, IL-17a, IFN-γ compared to the control group (P < 0.05). In conclusion, EVs derived from F. prausnitzii showed greater efficacy in decreasing the inflammatory cytokines and increasing the anti-inflammatory cytokines, compared to F. prausnitzii. Our findings can be used as a theoretical model for EVs application in the potential treatment of inflammation.