Diagnostic genosensor for detection of rotavirus based on HFGNs/MXene/PPY signal amplification.

A novel genosensor was developed for rotavirus specific cDNA sequence detection. The genosensor was comprised of hierarchical flower-like gold nanostructures, MXene, and polypyrrole (HFGNs/MXene/PPY) nanocomposite as a signal amplification tag, specific antisense ssDNA oligonucleotide as a recognition bioelement, and methylene blue (MB) as a redox marker. The morphological and electrochemical features of the biosensor were first tested and optimized and the high performance of the platform was confirmed in terms of sensitivity and reproducibility. Then, 20 rotavirus RNA isolated from clinical and cell-cultured samples (10 positive and 10 negative confirmed by RT-PCR and electrophoresis methods) were evaluated by the genosensor. The analysis results revealed that the genosensor is able to differentiate successfully between the positive and negative control groups. The developed genosensor for rotavirus RNA detection presented an excellent limit of detection of ∼ 0.8 aM and a determination  range of  10-18 and 10-7 M. In addition, the ssDNA/HFGNs/MXene/PPY/GCE showed high selectivity and long-term stability of ~ 24 days. Therefore, this novel genosensor would be of great benefit for the clinical diagnosis of rotavirus.

Rapid and sensitive detection of SARS-CoV-2 virus in human saliva samples using glycan based nanozyme: a clinical study.

A highly sensitive colorimetric method (glycan-based nano(e)zyme) was developed for sensitive and rapid detection of the SARS-CoV-2 virus based on N-acetyl neuraminic acid (sialic acid)-functionalized gold nanoparticles (SA-Au NZs). A number of techniques were used to characterize the prepared nanomaterials including XRD, FT-IR, UV-vis, DLS, and TEM. DLS analysis indicates an average hydrodynamic size of 34 nm, whereas TEM analysis indicates an average particle size of 15.78 nm. This observation confirms that water interacts with nanoparticle surfaces, resulting in a large hydrodynamic diameter. The peroxidase-like activity of SA-Au NZs was examined with SARS-CoV-2 and influenza viruses (influenza A (H1N1), influenza A (H3N2), and influenza B). UV-visible spectroscopy was used to monitor and record the results, as well as naked eye detection (photographs). SA-Au NZs exhibit a change in color from light red to purple when SARS-CoV-2 is present, and they exhibit a redshift in their spectrum. N-acetyl neuraminic acid interacts with SARS-CoV-2 spike glycoprotein, confirming its ability to bind glycans. As a result, SA-Au NZs can detect COVID-19 with sensitivity and specificity of over 95% and 98%, respectively. This method was approved by testing saliva samples from 533 suspected individuals at Ghaem Hospital of Mashhad, Mashhad, Iran. Sensitivity and specificity were calculated by comparing the results with the definitive results. The positive results were accompanied by a color change from bright red to purple within five minutes. Statistical analysis was performed based on variables such as age, gender, smoking, diabetes, hypertension, and lung involvement. In clinical trials, it was demonstrated that this method can be used to diagnose SARS-CoV-2 in a variety of places, such as medical centers, hospitals, airports, universities, and schools.

Mutations in SARS-CoV-2 structural proteins: a global analysis.

BACKGROUND: Emergence of new variants mainly variants of concerns (VOC) is caused by mutations in main structural proteins of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Therefore, we aimed to investigate the mutations among structural proteins of SARS-CoV-2 globally. METHODS: We analyzed samples of amino-acid sequences (AASs) for envelope (E), membrane (M), nucleocapsid (N), and spike (S) proteins from the declaration of the coronavirus 2019 (COVID-19) as pandemic to January 2022. The presence and location of mutations were then investigated by aligning the sequences to the reference sequence and categorizing them based on frequency and continent. Finally, the related human genes with the viral structural genes were discovered, and their interactions were reported. RESULTS: The results indicated that the most relative mutations among the E, M, N, and S AASs occurred in the regions of 7 to 14, 66 to 88, 164 to 205, and 508 to 635 AAs, respectively. The most frequent mutations in E, M, N, and S proteins were T9I, I82T, R203M/R203K, and D614G. D614G was the most frequent mutation in all six geographical areas. Following D614G, L18F, A222V, E484K, and N501Y, respectively, were ranked as the most frequent mutations in S protein globally. Besides, A-kinase Anchoring Protein 8 Like (AKAP8L) was shown as the linkage unit between M, E, and E cluster genes. CONCLUSION: Screening the structural protein mutations can help scientists introduce better drug and vaccine development strategies.

Development of detection methods for the diagnosis and analysis of highly toxic metal phosphides: A comprehensive and critical review.

Metal phosphides, especially aluminum phosphide, and phosphine (PH3 ) are widely used as insecticides and rodenticides for protection of grains during process of storage and transportation. The main reason of poisoning with this compound is related to the conscious ingestion of salts or accidental inhalation of PH3 . So the early and accurate diagnosis of poisoning can significantly help to the effective clinical treatment or recognition of death cause. PH3 is somewhat unstable due to reaction with oxygen or hemoglobin leading to formation of oxy-acids phosphorous. Here, we critically reviewed the literature introducing the quantitative and qualitative methods for the detection of metal phosphides, PH3 , and its products. This study obviously demonstrates that during past years, different diagnosis methods have been remarkably progressed. Head-space gas chromatography and confirmatory colorimetric methods have been as the most popular techniques. Also, the gas sensors are a promising method that must be more progressed.

Detection of Isoniazid and Rifampin Resistance in Mycobacterium tuberculosis Clinical Isolates from Sputum Samples by High-Resolution Melting Analysis.

The effective management of multidrug-resistant tuberculosis (MDR-TB) and the need for rapid and accurate screening of rifampin (RIF) and isoniazid (INH)-resistant Mycobacterium tuberculosis (Mtb) isolates are the most fundamental and difficult challenges facing the global TB control. The present study aimed to compare the diagnostic accuracy of high-resolution melting-curve analysis (HRMA) in comparison to multiplex allele-specific PCR (MAS-PCR) and xpert MTB/RIF as well as the conventional drug-susceptibility test (DST) and gene sequencing for the detection of INH and RIF resistance in the Mtb isolates. In the present study, a total of 431 Mtb isolates including 11 MDR (%2.55), 7 INH resistance (%1.62), two RIF resistance (%0.46), and 411 sensitive isolates were phenotypically confirmed. HRMA assay identified katG gene mutations and the mabA-inhA promoter region in 15 of 18 INH-resistant samples and rpoB gene mutations were successfully evaluated in 11 out of 13 RIF-resistant samples. The sensitivity and specificity of the HRMA method were 83.3% and 98.8% for INH and 84.6% and 99% for RIF, respectively. The most common mutation in RIF-resistance-determining region (RRDR) occurred at codon 531 (TCG → TTG)(84.6%) and then at codon 513 (CAA → GTA)(7.6%) and 526 (CAC → TAC) (7.6%), which resulted in the amino-acid changes. Also, 88.8% of INH-resistant samples had mutations in the katG gene and the mabA-inhA promoter region, of which the highest mutation occurred at codon 315 (AGC → ACC) of the katG gene. In conclusion, all these results indicated that the sensitivity and specificity of the HRM method were increased when the katG gene and the mabA-inhA promoter region were used as a target.

The overview and perspectives of biosensors and Mycobacterium tuberculosis: A systematic review.

Tuberculosis (TB) is referred to as a "consumption" or phthisis, which has been a fatal human disease for thousands of years. Mycobacterium tuberculosis (M. tb) might have been responsible for the death of more humans than any other bacterial pathogens. Therefore, the rapid diagnosis of this bacterial infection plays a pivotal role in the timely and appropriate treatment of the patients, as well as the prevention of disease spread. More than 98% of TB cases are reported in developing countries, and due to the lack of well-equipped and specialized diagnostic laboratories, development of effective diagnostic methods based on biosensors is essential for this bacterium. In this review, original articles published in English were retrieved from multiple databases, such as PubMed, Scopus, Google Scholar, Science Direct, and Cochrane Library during January 2010-October 2019. In addition, the reference lists of the articles were also searched. Among 109 electronically searched citations, 42 articles met the inclusion criteria. The highest potential and wide usage of biosensors for the diagnosis of M. tb and its drug resistance belonged to DNA electrochemical biosensors (isoniazid and rifampin strains). Use of biosensors is expanding for the detection of resistant strains of anti-TB antibiotics with high sensitivity and accuracy, while the speed of these sensory methods is considered essential as well. Furthermore, the lowest limit of detection (0.9 fg/ml) from an electrochemical DNA biosensor was based on graphene-modified iron-oxide chitosan hybrid deposited on fluorine tin oxide for the MPT64 antigen target. According to the results, the most common methods used for M. tb detection include acid-fast staining, cultivation, and polymerase chain reaction (PCR). Although molecular techniques (e.g., PCR and real-time PCR) are rapid and sensitive, they require sophisticated laboratory and apparatuses, as well as skilled personnel and expertise in the commentary of the results. Biosensors are fast, valid, and cost-efficient diagnostic method, and the improvement of their quality is of paramount importance in resource-constrained settings.

Immunogenicity of HspX/EsxS fusion protein of Mycobacterium tuberculosis along with ISCOMATRIX and PLUSCOM nano-adjuvants after subcutaneous administration in animal model.

BACKGROUND: Tuberculosis (TB), caused by Mycobacterium tuberculosis (M. tuberculosis), is one of the most common and dangerous infectious diseases in the world. Despite vaccination with BCG, it is still considered as a major health problem. Therefore, design and production of an effective novel vaccine against TB is necessary. Our aim was to evaluate immunogenicity of HspX/EsxS fusion protein of M. tuberculosis along with ISCOMATRIX, PLUSCOM nano-adjuvants and MPLA through the subcutaneous route in mice model. METHODS: HspX/EsxS fused protein of M. tuberculosis was cloned, expressed and purified in the prokaryotic system. ISCOMATRIX and PLUSCOM nano-adjuvants were prepared by film hydration method. Subcutaneous immunization of BALB/c mice was performed by different formulations. IFN-γ, IL-4, IL-17 and TGF-ß cytokines levels as well as serum IgG1, IgG2a. RESULTS: Our results showed that subcutaneous administration of mice with HspX/EsxS along with three adjuvants, ISCOMATRIX, PLUSCOM and MPLA increased immunogenicity of multi-stage fusion protein of M. tuberculosis. Additionally, HspX/EsxS protein + ISCOMATRIX or + PLUSCOM nano-adjuvants induced stronger Th1, IgG2a and IgG1 immune responses compared to MPLA adjuvant. Totally, HspX/EsxS/ISCOMATRIX/MPLA, HspX/EsxS/PLUSCOM/MPLA and two BCG booster groups could significantly induce higher Th1 and IgG2a immune responses. CONCLUSION: With regard to ability of ISCOMATRIX, PLUSCOM and MPLA adjuvants to increase immunogenicity of HspX/EsxS protein through induction of IFN-γ and IgG2a immune responses, it seems that these adjuvants and especially ISCOMATRIX and PLUSCOM, could also improve BCG efficacy as a BCG booster.

Rapid and label-free electrochemical DNA biosensor based on a facile one-step electrochemical synthesis of rGO-PPy-(L-Cys)-AuNPs nanocomposite for the HTLV-1 oligonucleotide detection.

Human T cell leukemia virus type 1 (HTLV-1) as the first human retrovirus is currently a serious endemic health challenge. Despite the use of assorted molecular or serological assays for HTLV-1 detection, there are several limitations due to the lack of a confirmatory test that may affect the accuracy of the results. Herein, a novel label-free biosensor for the detection of HTLV-1 Tax gene has been reported. An electrochemical facile ecofriendly synthesis method has been demonstrated based on a synthesis of nanocomposite of reduced graphene oxide, polypyrrole, and gold nanoparticles (rGO-PPy-(l-Cys)-AuNPs) deposited on the surface of screen-printed carbon electrode. Electrochemical techniques were used to characterize and study the electrochemical behavior of the rGO-PPy-(l-Cys)-AuNPs, which exhibited a stable reference peak at 0.21 V associated with hybridization forms by applying the differential pulse voltammetry. The designed DNA biosensor presented a wide linear range from 0.1 fM to 100 µM and a low detection limit of 20 atto-molar. The proposed biosensor presented in this study provides outstanding selectivity, sensitivity, repeatability, and reproducibility.

Nanotechnology-driven advances in the treatment of diabetic wounds.

Diabetic foot ulcers (DFUs) are chronic severe complications of diabetes disease and remain a worldwide clinical challenge with social and economic consequences. Diabetic wounds can cause infection, amputation of lower extremities, and even death. Several factors including impaired angiogenesis, vascular insufficiency, and bacterial infections result in a delayed process of wound healing in diabetic patients. Treatment of wound infections using traditional antibiotics has become a critical status. Thus, finding new therapeutic strategies to manage diabetic wounds is urgently needed. Nanotechnology has emerged as an efficient approach for this purpose. This review aimed to summarize recent advances using nanotechnology for the treatment of diabetic wounds.

Detection of efflux pump genes in multiresistant Acinetobacter baumannii ST2 in Iran.

Acinetobacter baumannii, as a nosocomial pathogen has become a worldwide concern in recent years. In the current study, the resistance to tetracyclines and colistin were assessed in the isolates from different provinces of Iran.During the timeline of this study, a number of 270 isolates of A. baumannii were collected from tracheal aspirates, wounds, urine and blood cultures. The minimum inhibitory concentration (MIC) for tetracycline, doxycycline, minocycline, tigecycline and colistin were evaluated. Tetracycline resistance genes were assessed by PCR. The mean expression level of adeB, adeJ and adeG were assessed using semi quantitative Real-Time PCR. The clonal relationship of the isolates was evaluated by the repetitive extragenic palindromic PCR (REP-PCR), International Clonal (IC) Lineage Multiplex PCR and multilocus sequence typing (MLST) (Pasteur scheme) methods.The MIC by microdilution method showed that 87.5, 51.4, 28, 0.74 and 0% of the isolates were resistant to tetracycline, doxycycline, minocycline, tigecycline and colistin respectively. The prevalence of tetracycline resistance genes was 99.2, 99.2, 98, 86.7, 10, 3.33, 0.37, 0% for adeB, adeJ, adeG, tetB, tetA(39), tetA, tetM and tetH in tetracycline-resistant isolates. Moreover, the expression level of adeB, adeJ, adeG genes in tigecycline-nonsusceptible A. baumannii (TNAB) strain was higher compared to the tigecycline-susceptible A. baumannii (TSAB). A broad genomic diversity was revealed, but ST2 was the most prevalent ST. Our results indicated that tetracycline resistance in Iran is mediated by resistance-nodulation-cell division (RND) and tetB efflux pumps.

Genotypic and phenotypic characterization of Mycobacterium tuberculosis resistance against fluoroquinolones in the northeast of Iran.

BACKGROUND: Fluoroquinolones are broad-spectrum antibiotics that are recommended, and increasingly important, for the treatment of multidrug-resistant tuberculosis (MDR-TB). Resistance to fluoroquinolones is caused by mutations in the Quinolone Resistance Determining Region (QRDR) of gyrA and gyrB genes of Mycobacterium tuberculosis. In this study, we characterized the phenotypic and genotypic resistance to fluoroquinolones for the first time in northeast Iran. METHODS: A total of 123 Mycobacterium tuberculosis isolates, including 111 clinical and 12 collected multidrug-resistant isolates were studied. Also, 19 WHO quality control strains were included in the study. The phenotypic susceptibility was determined by the proportion method on Löwenstein-Jensen medium. The molecular cause of resistance to the fluoroquinolone drugs ofloxacin and levofloxacin was investigated by sequencing of the QRDR region of the gyrA and gyrB genes. RESULTS: Among 123 isolates, six (4.8%) were fluoroquinolone-resistant according to phenotypic methods, and genotypically three of them had a mutation at codon 94 of the gyrA gene (Asp→ Gly) which was earlier reported to cause resistance. All three remaining phenotypically resistant isolates had a nucleotide change in codon 95. No mutations were found in the gyrB gene. Five of the 19 WHO quality control strains, were phenotypically fluoroquinolone-resistant, four of them were genotypically resistant with mutations at codon 90, 91 of the gyrA gene and one resistant strain had no detected mutation. CONCLUSIONS: Mutation at codon 94 of the gyrA gene, was the main cause of fluoroquinolone resistance among M. tuberculosis isolates in our region. In 3/6 fluoroquinolone-resistant isolates, no mutations were found in either gyrA or gyrB. Therefore, it can be concluded that various other factors may lead to fluoroquinolone resistance, such as active efflux pumps, decreased cell wall permeability, and drug inactivation.

Shedding light on the EpCAM: An overview.

The epithelial cell adhesion molecule (EpCAM) is a Type I transmembrane superficial glycoprotein antigen that is expressed on the surface of basolateral membrane of multiple epithelial cells with some exceptions such as epidermal keratinocytes, hepatocytes, thymic cortical epithelial cells, squamous stratified epithelial cells, and myoepithelial cells that do not express the molecule. The molecule plays a pivotal role in the structural integrity, adhesion of the epithelial tissues and their interaction with the underlying layers. EpCAM prevents claudin-7 and claudin-1 molecules from degradation, thereby, decreasing the number of tight junctions and cellular interconnections, and promoting the cells toward carcinogenic transformation. Moreover, the mutations in the EpCAM gene lead to congenital tufting enteropathy, severe intestinal epithelium homeostasis disorders, and Lynch and Lynch syndrome. Overexpression of EpCAM on stem cells of some cancers and the presence of this molecule on circulating tumor cells (CTCs) makes it a promising candidate for cancer diagnosis as well as tracing and isolation of CTCs.

Current approaches for detection of human T-lymphotropic virus Type 1: A systematic review.

BACKGROUND: Human T-lymphotropic virus Type 1 (HTLV-1) is a retrovirus that is endemic in some regions of the world. It is known to cause several diseases like adult T-cell leukemia (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Serology and molecular methods have been used to detect this virus. Of these, enzyme-linked immunosorbent assay (ELISA) is used as a primary screening method and this is usually followed by western blotting (WB) and polymerase chain reaction (PCR) methods as confirmatory tests. We conducted a systematic review of the different techniques used in the diagnosis of HTLV-1 infection. MATERIALS AND METHODS: Our search was limited to original papers in the English language from 2010 to 2018 using several databases including Pubmed, Scopus, Google Scholar, Iranmedex, and Scientific Information Database. A manual search of references provided in the included papers was also performed. RESULTS: Of 101 electronically searched citations, 43 met the inclusion criteria. ELISA is commonly used for qualitative and screening detection, and WB and PCR techniques are used to confirm infection. CONCLUSION: Among all the reported methods for detection of HTLV-1, only serological and molecular tests are used as the most common technical assays for HTLV-1. The ELISA assay, without a confirmatory test, has several limitations and affect the accuracy of the results. Owing to the prevalence of HTLV-1 and limitations of the current detection methods, further evaluation of the accuracy of these methods is needed. There are new opportunities for applying novel technological advances in microfluidics, biosensors, and lab-on-a-chip systems to perform HTLV-1 diagnostics.

Autophagy induction plays time-dependent role in viral load of HCV infected Huh7.5 cell line.

Autophagy provides an initial membranous platform for incoming hepatitis C virus (HCV) RNA translation and immune evasion. Once HCV replication is established, this infrastructure will be unnecessary for translation of HCV RNA progeny. So, the autophagy plays key role in the replication and immune pathogenesis of HCV virus. The aim of this study was to study the effect of autophagy induction in Huh7.5 cell on virus titer. The Huh7.5 cell was transfected with recombinant pcDNA-Beclin1. The autophagy induction was evaluated via microtubule associated protein 1 light chain 3 staining as autophagy formation marker using flow cytometry. The HCV (JFH1) was inoculated 12-h post-transfection. Next, to evaluate the viral load, viral RNA was extracted after 24 and 48 h and virus titer was calculated using real-time PCR. The result of the current study shows that the induction of autophagy before virus infection was able to enhance virus yield from 4 × 103 copies/mL to 1 × 104 copies/mL at 24-h post-infection, but reduced viral load after 48 h up to 6 × 103 copies/mL. The study of cross-talk between autophagy and HCV may bring new hope for human intervention and treatment of HCV. Also, it opens new avenue to improve virus cultivation in cell culture and understanding HCV and host cell responses. © 2018 IUBMB Life, 71(1):41-44, 2019.

Prevalence of CCR5delta32 in Northeastern Iran.

BACKGROUND: A 32-base pair deletion (∆32) in the open reading frame (ORF) of C-C motif chemokine receptor 5 (CCR5) seems to be a protective variant against immune system diseases, especially human immunodeficiency virus type 1 (HIV-1). We aimed to assess the frequency of CCR5∆32 in the healthy Iranian population. METHODS: In this study, 400 normal samples from Khorasan, northeastern Iran, were randomly selected. The frequency of CCR5∆32 carriers was investigated using PCR analysis. Allele prevalence and the fit to the Hardy-Weinberg equilibrium were analyzed. RESULTS: The prevalence of CCR5∆32 in the northeastern population of Iran was 0.016. Four hundred samples were studied, among which one with CCR5∆32/∆32 and 11 with CCR5Wild/∆32 genotype were detected. CONCLUSION: This study was the first investigation for an assessment of the prevalence of CCR5∆32 in northeastern Iran. The low prevalence of CCR5∆32 allele in the Iranian population may result in the increased susceptibility to HIV-1. In addition, this prevalence is the same as that of reported in East Asia, while is lower than that in the Europeans.

A method for improving the efficiency of DNA extraction from clotted blood samples.

BACKGROUND: The efficient and rapid extraction of high-quality genomic DNA from clotted blood samples, which normally have a low yield and poor quality, is an important factor in genomic research. The objective of this study was to develop a simple and safe technique for dispersing the blood clots by the ball bearing metal shots. Normally, such clot samples may not have an acceptable yield by conventional DNA extraction methods. Also, in the present study, we have further investigated to improve salting-out DNA extraction methods. METHODS: Initially, 500 µL phosphate-buffered saline (PBS) (1×) and two ball bearing metal shots were added to each tube of the clotted blood sample and then were gently rotated in an electric laboratory rotator for 1 hour at room temperature (18-25°C). Genomic DNA was then extracted from samples using a modified salting-out method and a modified QIAamp® DNA Blood Midi Kit and was compared with QIAamp® DNA Blood Midi Kit as a control. An assessment of the concentration and quality of the extracted DNA was performed using the UV-visible spectrophotometer. The isolated DNA proved amenable to PCR amplification and gel electrophoresis. RESULTS: The yield and purity of DNA obtained by these three methods were significantly different (P < 0.001), with a higher yield in the modified salting-out method. CONCLUSIONS: Our proposed modified salting-out method is simple and efficient for the isolation of DNA from old blood clot samples. It is both easy to use and is of low cost in routine laboratory tasks.

The efficacy of vaginal suppository based on myrtle in patients with cervicovaginal human papillomavirus infection: A randomized, double-blind, placebo trial.

Human papillomavirus (HPV) can be detected in most of cervical cancers. Due to antiviral, antimutagenic, and proapoptotic activities of myrtle, this study was designed to investigate the effect of a herbal suppository based on myrtle in cervicovaginal HPV infections. This study was performed as a double-blind randomized trial at the Clinic of Traditional Medicine in Mashhad University of Medical Sciences between 2016 and 2017. Sixty women, 18 to 50 years old, with cervicovaginal HPV infection, were included and randomly allocated to two groups. Sixty placebo or herbal vaginal suppositories were prescribed for 3 months (20 suppositories at each menstrual cycle). Each herbal vaginal suppository contained 10% of myrtle aqueous extract and 0.5% of myrtle essential oil. The HPV test and colposcopic findings were evaluated after treatment. There was no difference between two groups as regards lesion site, diagnosis time of disease, and HPV type before intervention (p ≥ 0.05). At the end of the study, the HPV test was negative in 92.6% and 62.6% of the intervention and placebo groups, respectively (p = 0.036). The change in cervical lesion size was 71.4% and 30.4% in the intervention and placebo groups, respectively, based on colposcopic findings (p = 0.015). It seems that herbal suppository can speed up virus clearance and can be effective in treating HPV infection.

Molecular characterization and genetic relatedness of clinically Acinetobacter baumanii isolates conferring increased resistance to the first and second generations of tetracyclines in Iran.

BACKGROUND: The increasing resistance of Acinetobacter baumannii to antibiotics has recently been regarded as a notable therapeutic difficulty. Evaluating resistance rates of some A. baumannii isolates to tetracyclines had an impact on understanding the antibiotic resistance dissemination. By comparing genetic characteristics and relatedness of A. baumannii isolates, we are able to determine the transition dynamics of outbreak isolates. METHODS: A total of 72 non-duplicate isolates of A. baumannii were recovered in 2011 and 2015 and minimum inhibitory concentration (MIC) range distribution of the isolates to tetracyclines was performed by broth micro dilution (BMD) assay, and to determine the lineage relatedness of the outbreak isolates repetitive extragenic palindromic element based on polymerase chain reaction (rep-PCR) and international clonal (ICs) investigations were performed. RESULTS: Resistance rates to tetracycline, doxycycline and minocycline in 2011 were 73, 2 and 0%, while these rates in 2015 increased up to 90, 84 and 52%, respectively. The tetB existed in 100% of all the isolates of both years. tetA was not found in any of the isolates. According to the rep-PCR assays, up to 83% of all isolates clustered distinctly and only 6% of isolates had a common root. The percentage rates of IC1 decreased from 42% in 2011 to 22% in 2015, while those of IC2 increased from 28 to 36%, from 2011 to 2015. CONCLUSIONS: Our data showed that resistance to the first and second generations of tetracyclines is on the rise and the clonal transition dynamics of isolates are in progress in our hospital.

Construction and immunogenicity of a new Fc-based subunit vaccine candidate against Mycobacterium tuberculosis.

As an ancient disease, tuberculosis (TB) is a major global health threat. Therefore, there is an urgent need for an effective and safe anti-TB vaccine. In the current study, a delivery system of Fc domain of mouse IgG2a and early secreted antigenic target protein 6 (ESAT-6) was evaluated for the selective uptake of antigens by antigen-presenting cells (APCs). Thus, it was based on the immunogenicity of a fusion protein. The study was initiated by the transfer of recombinant expression vectors of pPICZαA-ESAT-6:Fcγ2a and pPICZαA-ESAT-6: His into Pichia pastoris (P. pastoris). Recombinant proteins were assessed for immunogenicity following the immunoblotting analysis. High levels of IFN-γ and IL-12 were produced to induce Th1-type cellular responses through vaccination with both recombinant proteins [ESAT-6:Fcγ2a (EF) and ESAT-6:His (EH)]. The Fc-tagged recombinant protein induced more effective Th1-type cellular responses with a low increment in IL-4 compared to PBS, BCG, and EH groups. Although in all the immunized groups, the ratio of IFN-γ/IL-4 was in favor of Th1 responses, the highest Th1/Th2 balance was observed in EF immunized group. Fc fragment of mouse IgG2a may induce a selective uptake of APCs towards the cross-presentation and formation of Th1 responses in favor of an appropriate protective anti-tuberculosis reaction. Thus, further research on Fc-fusion proteins is required to develop Fc-based TB vaccines.

Cytokine and growth factor profiling in patients with the metabolic syndrome.

The metabolic syndrome (MetS) is associated with a pro-inflammatory milieu that may partially account for its association with an increased cardiovascular risk. We aimed to (1) evaluate the serum concentrations of twelve cytokines and growth factors (epidermal growth factor (EGF), interferon-γ (IFN-γ), IL-1α/-1ß/-2/-4/-6/-8/-10, monocyte chemoattractant protein-1 (MCP-1), TNF-α and vascular endothelial growth factor (VEGF)) in 303 individuals with or without the MetS; and (2) explore their relationship with the presence of the MetS. Patients with the MetS had significantly higher serum concentrations of IFN-γ, EGF, IL-1α/-1ß/-2/-4/-6/-8/-10, MCP-1 and TNF-α, whilst serum VEGF concentrations were markedly lower compared with the control group (e.g. 38·55 v. 82·18 pg/ml; P< 0·05). Amongst these parameters, IFN-γ and IL-1α emerged as the most significant independent predictors of the MetS. In conclusion, our findings demonstrate that patients with the MetS had an altered blood cytokine and growth factor profile that may partially account for its adverse clinical outcomes. Further prospective studies in larger multi-centre settings are required to unravel the role and association of the emerging biomarkers with the MetS and their implication in therapeutic intervention.