mycobacterium tuberculosis

Tuberculosis (TB) remains a global health challenge, particularly due to drug resistance and limitations in rapid diagnosis. Next-generation sequencing (NGS), especially long-read whole genome sequencing (WGS), shows promise for rapidly detecting TB and drug resistance, but it requires high-quality DNA, which is difficult to extract from Mycobacterium tuberculosis due to its complex cell wall.

This study evaluated four DNA isolation methods for extracting pure DNA from M. tuberculosis , aiming to standardize protocols for long-read WGS.

Mycobacterium tuberculosis H37RV colonies were grown in BACTEC MGIT liquid medium. Two pellets were prepared as the initial material for the DNA extraction protocol: Pellets from 1 mL McFarland 2 suspensions and all growing colonies from two MGIT liquid cultures. Four DNA extraction methods were used: The cetyltrimethylammonium bromide (CTAB) method, GeneJET Genomic DNA Purification Kit, Quick-DNA Fecal/Soil Microbe Kit, and Genematrix Tissue/Bacterial DNA Purification Kit, with some modifications. DNA quality was assessed based on concentration, purity, and integrity.

Among the tested methods, the Quick-DNA Fecal/Soil Kit yielded approximately 85 ng/mL of DNA and a purity of 1.9 at 260/280 nm from the colonial pellet of two MGIT tubes. However, lower intact DNA [DNA integrity number (DIN) ~ 6.8] was obtained with this kit. The CTAB method provided the highest intact DNA (DIN ~ 9.5), although the purity of the DNA was not sufficient.

Based on three repetitions of McF-2 and colonial pellet extractions, the Quick-DNA Fecal/Soil Kit yielded the highest DNA quantity and purity but showed lower integrity compared to other methods, indicating the need for adjustments. A pellet from two MGIT cultures (~ 100 µL) is suitable for long-read WGS with this kit. However, a larger sample size is required to generalize these findings. For effective long-read sequencing of M. tuberculosis , DNA extraction protocols must be optimized to balance yield, fragment size, and purity for accurate sequencing and drug resistance analysis.

Despite the reduction of tuberculosis globally, new cases of this disease continue to be reported in Iran, although skin tuberculosis is uncommon and constitutes a small portion of new cases. The presentation of skin ulcers caused by cutaneous tuberculosis can mimic other systemic diseases, leading to clinical uncertainty. Cutaneous tuberculosis is classified into two types: the true type, characterized by the presence of bacilli in the affected skin, and the tuberculoid type, resulting from an allergic reaction.This case report highlights a situation where a skin nodule, initially identified as cutaneous tuberculosis, was later confirmed through a lung biopsy guided by CT scan. It emphasizes the significance of medical history, physical examination, and considering the potential risk of disease spread during needle sampling procedures guided by CT scans.

TB infection is one of the most challengeable epidemiological issues. Complex interactions between microbiota and TB infection have been demonstrated. Alteration in microbial population during TB infection may act as a useful biomarker. The present study examined the microbiota patterns of blood and sputum samples collected from Afghan immigrants and Iranian patients with active TB.

Sixty active pulmonary TB patients were enrolled in the study. Blood and sputum samples were collected. To detect phylum bacterial composition in the blood and sputum samples, bacterial 16S rRNA quantification by Real-Time qPCR was performed.

A significant decrease in Bacteroidetes in Iranian sputum and blood samples of Afghan immigrants and Iranian TB active subjects were seen. While, sputum samples of Afghan immigrants showed no significant differences in Bacteroidetes abundance among TB active and control. Firmicutes were also presented no significant difference between sputum samples of the two races. Actinobacteria showed a significant increase in Iranian and Afghan sputum samples while this phylum showed no significant abundance in Iranian and Afghan TB positive blood samples. Proteobacteria also showed an increase in sputum and blood samples of the two races.

An imbalance in Bacteroidetes and Firmicutes abundance may cause an alteration in the microbiota compo- sition, resulting in dysregulated immune responses and resulting in the augmentation of opportunistic pathogens during TB infection, notably Proteobacteria and Actinobacteria. Evaluation of human microbiota under different conditions of TB infection can be critical to a deeper understanding of the disease control.

Tuberculosis (TB) has been a major health issue throughout history. As part of TB infection, host-Mycobacterium tuberculosis (Mtb) interactions are important. Through immune pathology and cell death control processes, Mtb infection facilitates intracellular growth. The relationship between apoptosis and inflammation in Mtb infection remains unclear. In this study, the levels of related apoptosis and inflammatory genes were assessed in A549 cells infected with a variety of Mtb strains.

Mtb isolates with different phenotypes (sensitive, INHR, RifR, MDR, and XDR) were collected from the Pasteur Institute of Iran, during this study. Whole genome sequencing was previously performed on all strains, and the Beijing genotype was selected as sensitive. Also, for other resistant strains, the New-1 genotype was available and isolated for genotype comparison. A549 lung carcinoma cells were also grown and infected with selected Mtb strains. Genes involved in inflammation and apoptosis were detected using reverse transcription-PCR (RT-PCR).

All sensitive strains and resistant strains were found to significantly up-regulate anti-apoptotic (bcl2 and rb1), chemokine (IL-8 and MCP-1), and pro-inflammatory cytokine (TNF-α and IFN-γ) expression, while significant down-regulation was observed after 24 and 48 hr of infection in anti-inflammatory genes (IL-10) and pro-apoptotic genes (bad and bax). Besides resistance strains, Mtb genotypes also affected gene expression. The Beijing genotype (sensitive isolate) influences inflammatory and apoptotic genes more sharply than the New-1 genotype (INHR, RifR, MDR, and XDR).

Gene expression differences related to apoptosis and inflammation examined in the current study may be attributed to genotypes rather than resistance status since the expression of most genes has been observed to be lower in resistant strains (INHR, RifR, MDR, and XDR belonging to the New-1 genotype) compared to sensitive strains (Beijing genotype).

Tuberculosis (TB) is one of the most common infectious diseases in the world and requires novel medications or existing ones should be improved.  Nanotechnology is a modern science that helps to avoid adverse reactions and resistance to drugs. The current regimen for standard therapy calls for routine administration of medications over six months. Since the noncompliance of patients and the emergence of drug-resistant strains, therapies become more challenging. The objective of the current study was to develop Isoniazid-Rifampicin-loaded (INH-RIF-NPs) nanoparticles to improve release properties and drug encapsulation efficiency.

Box-Behnken Design (BBD) was used for optimizing the nanoparticles. Eudragit was used in the preparations in varying concentrations (1-2% w/v). The compatibility of the drug and excipients was shown. The existence of the nanoparticles was confirmed by the analytical results of the transmission electron microscopy (TEM) and Fourier transform infrared spectroscopy (FTIR). 

The optimized nanoparticles showed no drug-polymer interaction. The mean size of the INH-RIF-NPs was around 112±8.73 nm, and they were sphere-like, smooth, fairly uniform in size, and well-dispersed, and entrapment efficiencies were high at 98.7±0.68%. Drug release was slow and sustained with 66.91% INH cumulative release and 80.06 of RFP after 24 hr. 

Significant drug uptake with higher encapsulation efficiency, uniform size, good dispersion, and prolonged release characteristics are all present in INH-RIF-NPs. This suggests the existence of a delivery system capable of effectively encapsulating and delivery of combined drug formulation in polymeric nanoparticles.

 Isoniazid (INH) is one of the first-line drugs used for the treatment of Mycobacterium tuberculosis (Mtb). This study aimed to determine the mutations in katG and inhA promoter regions associated with INH resistance in clinical isolates of Mtb from Tehran, Iran.

 This descriptive cross-sectional study was conducted in the tuberculosis (TB) center of Tehran in 2020. 50 samples obtained from these patients were cultured on Löwenstein-Jensen medium (LJ), then, INH-resistant strains and their minimum inhibitory concentration (MIC) were determined using the proportional method. To determine the INH-resistance mutations, sequencing was performed following the amplification of both katG and inhA genes using real-time polymerase chain reaction (PCR). The genetic relationships were determined based on each strain's genetic pattern using the primers related to enterobacterial repetitive intergenic consensus PCR (ERIC-PCR), which is a proper tool used for typing Mtb strains. In this study, the phylogenetic tree was plotted using NTSYS software.

 Five INH-resistant Mtb strains were isolated from 50 patients with TB in Tehran. All the studied resistant strains (100%) showed a mutation in codon 315 of the katG gene; none of them exhibited any mutation in the promoter. I335T mutation was observed in one INH-resistant strain (20%). The phylogenetic tree of the strains indicated seven clusters as well as 31 patterns in the strains. The strain with two mutations in 335 and 315 had MIC > 8.

 KatG mutation could result in a high level of INH resistance. Therefore, routine identification of this mutation can help in determining the INH resistance, thereby preventing further propagation of these strains.

 Indonesia has the second highest tuberculosis (TB) cases globally. This study aimed to determine the sociodemographic factors associated with TB and rifampicin-resistant tuberculosis (RR-TB) cases among presumptive pulmonary TB patients in Aceh Referral Hospital.

Study Design:

 A retrospective cross-sectional study.

 A retrospective cross-sectional review of presumptive pulmonary TB patients having a sputum test at the clinical microbiology laboratory was conducted from January 2015 to December 2021. Patient characteristics and drug susceptibility data were abstracted from the hospital information system of TB (SITB) and analyzed by univariate and bivariate analysis.

 The Mycobacterium tuberculosis (MTB) was detected in 32.8% sample (1,521/4,637). Of the TB-confirmed cases, 14.1% (215/1,521) were resistant to rifampicin (RR-TB). Most of them were male patients (71.63%), were in the age range of 35–54 years (48.7%), lived in rural areas of the country (56.3%), and were previously TB-treated cases (65.5%). Overall, 35–44-year-old patients (adjusted odds ratio [AOR]=2.11, 95% CI=1.25, 3.5, P<0.05) were more likely to have RR-TB compared to>65-year-old patients. Gender and residence were not associated with RR-TB (P>0.05). Case detection decreased in pandemic conditions (19.5% in 2019 to 13.9% and 7.91% in 2020 and 2021, respectively).

 The findings revealed the dynamic cases and sociodemographic factors of TB and RR-TB in a province referral hospital in Indonesia for 7 years. The cases of TB and RR-TB among presumptive TB patients were 32.8% and 14.1%, respectively. The cases were found to be more noticeable in males, adults (45–54 years old), and patients residing in rural areas.

Mycobacterium tuberculosis is still one of the most dangerous human pathogens. Identification of the relationships between different clinical strains has remained a high priority for epidemiology research.

In this study, we used MLSA (Multilocus sequence analysis) to generate a highly robust phylogeny of M. tuberculosis. MLSA, based on single nucleotide polymorphism (SNP) was performed on five genes fragments from the Rpsl (302 bp), MprA (559 bp), LipR (322 bp), KatG (488 bp) and Fgd1 (266 bp), in order to identify polymorphic nucleotide sites, and the discriminatory power of each locus for all genes was measured with Hunter‐Gaston Index (HGI).

In this study, a sequence type (ST) number was assigned to each unique allelic profile, and 9 sequence types were identified from 20 strains, these imply that there is a high diversity of strains in this area.

Our results showed that the presence of high genetic diversity among clinical isolates of M. tuberculosis in Northeast of Iran. There is no evidence for recent transmission. Keywords: Mycobacterium tuberculosis, Multi-locus sequence analysis; Molecular epidemiology; Tuberculosis; KatG; Rpsl1. IntroductionMycobacterium tuberculosis (M. tuberculosis), the causative agents of tuberculosis (TB), is one of the most successful human pathogens, infecting nearly one-third of the people all around the world, causing over 9 million new cases and 1.7 million deaths each year [1-2]. Identification of the relationships between different clinical strains of M. tuberculosis has great significance to the public health [3].

Tuberculosis (TB) is a chronic granulomatous disease, ranking as the second cause of death by infectious agents in 2021. Tuberculosis affects millions worldwide, with valvular TB being a rare manifestation, accounting for less than 1% of extrapulmonary TB cases. Valvular TB is often diagnosed through imaging and laboratory evaluations, and the treatment involves prolonged antibiotic therapy and might require surgical intervention for damaged cardiac valves.

A 60-year-old female patient with diabetes mellitus and rheumatoid arthritis was referred for a cardiac assessment prior to hand wound debridement surgery. Transthoracic echocardiography (TTE) revealed a mass on the mitral valve, and subsequent transesophageal echocardiography (TEE) confirmed a large, mobile mass with abscess-like density. A blood culture study was negative for infective endocarditis, and a biopsy of the wound revealed necrotizing granuloma positive for Mycobacterium tuberculosis. Anti-TB medication was initiated, resulting in a significant improvement in the valve mass. No cardiac complications were reported during the follow-up.

In suspected cardiac TB patients with a high risk of surgery or patients reluctant to undergo an operation, quadruple chemotherapy can0 serve as a diagnostic and therapeutic tool.

Tuberculosis (TB), a contagious disease caused by Mycobacterium tuberculosis  (M. tuberculosis), remains a health problem worldwide and this infection has the highest mortality rate among bacterial infections. Current studies suggest that intranasal administration of new TB vaccines could enhance the immunogenicity of M. tuberculosis antigens. Hence, we aim to evaluate the protective efficacy and immunogenicity of HspX/EsxS fusion protein of M. tuberculosis along with ISCOMATRIX and PLUSCOM nano-adjuvants and MPLA through intranasal administration in a mice model.

In the present study, the recombinant fusion protein was expressed in Escherichia coli and purified and used to prepare different nanoparticle formulations in combination with ISCOMATRIX and PLUSCOM nano-adjuvants and MPLA. Mice were intranasally vaccinated with each formulation three times at an interval of 2 weeks. Three weeks after the final vaccination, IFN-γ, IL-4. IL-17, and TGF-β concentrations in the supernatant of cultured splenocytes of vaccinated mice as well as serum titers of IgG1 and IgG2a and sIgA titers in nasal lavage were determined.

According to obtained results, intranasally vaccinated mice with formulations containing ISCOMATRIX and PLUSCOM nano-adjuvants and MPLA could effectively induce IFN-γ and sIgA responses. Moreover, both HspX/EsxS/ISCOMATRIX/MPLA and HspX/EsxS/PLUSCOM/MPLA and their BCG booster formulation could strongly stimulate the immune system and enhance the immunogenicity of M. tuberculosis antigens.

The results demonstrate the potential of HspX/EsxS-fused protein in combination with ISCOMATRIX, PLUSCOM, and MPLA after nasal administration in enhancing the immune response against M. tuberculosis antigens. Both nanoparticles were good adjuvants in order to promote the immunogenicity of TB-fused antigens. So, nasal immunization with these formulations, could induce immune responses and be considered a new TB vaccine or a BCG booster.

Tuberculosis, caused by Mycobacterium tuberculosis, is one of the most common infectious diseases worldwide. Epidemiological studies of M. tuberculosis drug resistance are critical for improving patient treatment approaches and controlling the spread of tuberculosis. The present study aimed to determine antibiotic resistance among M. tuberculosis clinical isolates using the Microplate Alamar Blue Assay (MABA).

In this descriptive cross-sectional study, 25 M. tuberculosis isolates from clinical samples were identified and confirmed using standard microbiological and biochemical tests. Then, the MIC for the antibiotics Bedaquiline, isoniazid, rifampin, ethambutol, ofloxacin, moxifloxacin, capreomycin, and streptomycin was determined using the MABA method. The results were analyzed using SPSS version 16 software.

Among the 25 investigated isolates, the frequencies for MDR, Pre-XDR, and XDR isolates were 20%, 8%, and 32%, respectively. The highest rate of drug resistance was to isoniazid (80%), rifampicin, and ethambutol (76%), and the highest rate of sensitivity was to moxifloxacin (68%). The frequency of isoniazid mono-resistance and rifampicin mono-resistance was 5 cases (50%) and 4 cases (40%), respectively.

Our study revealed an alarming rate of MDR and XDR M. tuberculosis strains, indicating that current first-line treatments may be ineffective for a significant number of patients. The bedaquiline resistance among the isolates with no history of previous exposure to this drug suggests unexplored resistance mechanisms. Molecular techniques to accurately identify these mechanisms may contribute to developing more effective treatment strategies to combat drug-resistant tuberculosis.

Tuberculosis (TB) caused by the bacterium Mycobacterium tuberculosis remains a critical global public health concern due to the high morbidity and mortality rates. Mutation in atpE and Rv0678 genes contributes to drug resistance in M. tuberculosis. This study investigates the antibiotic resistance patterns and mutations in atpE and Rv0678 genes in 22 M. tuberculosis clinical isolates.

Drug susceptibility testing (DST) for rifampin, isoniazid, streptomycin, capreomycin, ofloxacin, kanamycin, and ethambutol was conducted using the proportional method. This was followed by determining the minimum inhibitory concentration (MIC) for bedaquiline (BDQ) via the microplate Alamar blue assay (MABA). Genomic regions encompassing atpE and Rv0678 genes were amplified and sequenced for mutation analysis. Data analysis was performed using SPSS software to interpret mutation patterns concerning drug susceptibility profiles.

Of 22 isolates, 5 (27.8%) were extensively drug-resistant tuberculosis (XDR-TB), and 13 (72.2%) were multi-drug resistant tuberculosis (MDR-TB). Resistance rates to kanamycin, ofloxacin, capreomycin, and streptomycin were 40.6%, 46.3%, 85%, and 74.6%, respectively. Additionally, phenotypic resistance to bedaquiline was observed in 12 (54.5%) isolates. Sequencing revealed no resistance-conferring mutations in the atpE or Rv0678 genes among the isolates.

Our findings showed substantial resistance to first- and second-line drugs in M. tuberculosis clinical isolates. This highlights the necessity for ongoing, comprehensive studies to elucidate the evolving drug resistance patterns and understand the underlying mechanisms in clinical isolates.

The role of microRNAs (miRNAs) in tuberculosis infection is well established. As microR- NAs are able to change expression profiles according to different conditions, they can be useful biomarkers. Iranians and Afghans with tuberculosis were studied for three immune-related miRNAs (miR-let-7f, miR-125a, and miR-125b).

A total of 60 Iranian and Afghan patients with active pulmonary TB were enrolled in the Pulmo- nary Department of the Pasteur Institute of Iran. Serum and sputum samples were collected simultaneously from all partici- pants. A Real-time PCR was conducted to detect differentially expressed miRNAs.

Iranian (P<0.0001) and Afghan (P<0.0001) serum samples and Afghan (P<0.0001) sputum samples overexpressed miR-125a, whereas Iranian sputum samples showed downregulation (P=0.0039). In both Iranian (P<0.0001; P=0.0007) and Afghan (P<0.0001; P<0.0001) serum and sputum samples, miR-125b was overexpressed. Furthermore, miR-let-7f down- regulation was observed in serum and sputum samples (P<0.0001), whereas Iranian sputum samples had no statistically significant differences (P=0.348).

Overexpression of miR-125a and miR-125b has been detected in Iranian and Afghan samples. In both races, miR-let-7f downregulation has been confirmed. Identification of miRNA profiles under different conditions opens the door to evaluating potential new biomarkers for diagnosis, disease monitoring, and therapeutic markers in TB infection.

Latent tuberculosis infection (LTBI) prevalence varies dramatically by region, with one-fourth of the world's population infected. The number of people with LTBI progressing to active tuberculosis illness (aTB) should be decreased to meet the WHO End TB Strategy objective of decreasing worldwide tuberculosis incidence by 2030. Current tuberculosis (TB) diagnostic methods are based on detecting an immune response to mycobacterial antigens injected into the skin or in vitro simulations in interferon-gamma release assays. Both tests have low sensitivity, which cannot distinguish between LTBI and aTB. Therefore, various techniques, such as alternate cytokine detection and employing novel antigens, are being studied to increase the accuracy of these tests.  In addition, novel antigens can be used to monitor aTB progression and response to treatment. This review aims to assess current available diagnostic tools and evidence on novel Mycobacterium tuberculosis (Mtb) antigens for LTBI diagnosis and select the most promising antigens for future research.