A Comparative Study on Visual Detection of Mycobacterium tuberculosis by Closed Tube Loop-Mediated Isothermal Amplification: Shedding Light on the Use of Eriochrome Black T.

Loop-mediated isothermal amplification is a promising candidate for the rapid detection of Mycobacterium tuberculosis. However, the high potential for carry-over contamination is the main obstacle to its routine use. Here, a closed tube LAMP was intended for the visual detection of Mtb to compare turbidimetric and two more favorable colorimetric methods using calcein and hydroxy naphthol blue (HNB). Additionally, a less studied dye (i.e., eriochrome black T (EBT)) was optimized in detail in the reaction for the first time. Mtb purified DNA and 30 clinical specimens were used to respectively determine the analytical and diagnostic sensitivities of each method. The turbidimetric method resulted in the best analytical sensitivity (100 fg DNA/reaction), diagnostic sensitivity and specificity (100%), and time-to-positivity of the test (15 min). However, this method is highly prone to subjective error in reading the results. Moreover, HNB-, calcein-, and EBT-LAMP could respectively detect 100 fg, 1 pg, and 1 pg DNA/reaction (the analytical sensitivities) in 30, 15, and 30 min, while the diagnostic sensitivity and specificity were respectively 93.3% and 100% for them all. Interestingly, EBT-LAMP showed the lowest potential for subjective error in reading the results. This report helps judiciously choose the most appropriate visual method, taking a step forward toward the field applicability of LAMP for the detection of Mtb, particularly in resource-limited settings.

Clinical Evaluation of Commercial HARDSON COVID-19 Antigen Rapid Test Kit for Routine COVID-19 Diagnosis.

Objective: This study aimed to evaluate the sensitivity, specificity, and accuracy of the commercial HARDSON COVID-19 Antigen Rapid Test Kit for diagnosing COVID-19 among the Iranian population by compared with the results of commercial RT-PCR. Materials and Methods: Two nasopharyngeal swabs were collected from each patient. One swab was tested with HARDSON COVID-19 Antigen Rapid Test Kit, and the second swab was placed in 3 mL of a virus-transmitted inactivated media for RT-PCR testing. Then, the results of both tests were compared to investigate the diagnostic accuracy of the rapid antigen test. Results: A total of 275 suspected COVID-19 patients' samples were collected to investigate the diagnostic accuracy of HARDSON COVID-19 Antigen Rapid Test Kit. In this study, 162 positive and 113 negative samples were evaluated. As a result, the sensitivity, specificity, and accuracy of HARDSON COVID-19 Antigen Rapid Test Kit were 90%, 100%, and 94%, respectively. Conclusion: The diagnostic kit analyzed in this study indicated excellent specificity and a relatively good overall sensitivity for the diagnosis of COVID-19 when compared with the RT-PCR detection kit. Based on the result of this study, COVID-19 Antigen Rapid Test Kit indicated a good sensitivity (96%) in low cycle threshold (Ct) value, and it would be recommended to be integrated into routine diagnostic laboratories and used as an at-home rapid antigen test.

A New Specific DNA Target Sequence for Identification of Staphylococcus epidermidis using Modified Comparative Genomic Analysis.

Background: Staphylococcus epidermidis (S. epidermidis) is the most frequently isolated pathogen from prostheses infections in the body. Therefore, improving its diagnostic methods, including rapid Nucleic Acid Amplification Tests (NAAT), seems necessary. Since the first step in designing a NAAT is to find a specific sequence and all DNA targets that have been introduced so far are not completely specific, we introduced a new 100% specific DNA target sequence to identify S. epidermidis in this study. Methods: Modified comparative genomic analysis was used to find the best specific target sequence to detect S. epidermidis. A PCR method was designed for the evaluation of this target. To determine the detection limit and analytical specificity, pure genomic DNA of 18 bacteria include 12 standard strains (one S. epidermidis and 11 non-S. epidermidis) and six clinical isolates (five S. epidermidis and one non-S. epidermidis) were used. Results: The 400 bp sequence of S. epidermidis ATCC 14990 was identified as the most specific sequence (Se400), having a 100% sequence similarity to S. epidermidis genomes but not with other bacteria. The detection limit of Se400-PCR was 10 fg, equal to about 4 copies of S. epidermidis genomic DNA/µl. All pure DNA templates from S. epidermidis generated a detectable amplicon by 264 bp length, but the PCR test was negative for the non-S. epidermidis group. Conclusion: The Se400 sequence can be considered as a specific target for detecting S. epidermidis, based on our findings.

The first diagnostic test for specific detection of Mycobacterium simiae using an electrochemical label-free DNA nanobiosensor.

Mycobacterium simiae has been reported to be the most prevalent species of Nontuberculous mycobacteria (NTM) in many countries. As both phenotypic and molecular detection of M. simiae and other NTMs have limitations, finding an accurate, fast, and low-cost diagnostic method is critical for the management of infections. Here, we report the development of a new type of label-free electrochemical biosensor using a gold electrode decorated with l-cysteine/PAMAM dendrimer for specific targeting of M. simiae ITS sequence. DNA hybridization was monitored by measuring changes in the free guanine electrical signal with changing ssDNA target concentrations by differential pulse voltammetry (DPV) method. Response surface methodology (RSM) was applied for the optimization of variables affecting biosensor response. Under optimal conditions, the biosensor revealed a wide linear range from 10-14 M to 10-6 M and a detection limit of 1.40 fM. The fabricated biosensor showed an excellent selectivity to M. simiae in the presence of other similar pathogenic bacteria. Moreover, experimental results confirmed that this biosensor exhibited great precision and high reproducibility, hence provides a low-cost, label-free, and faster detection analysis, representing a novel strategy in detecting other NTMs.

Synergistic effect of two antimicrobial peptides, Nisin and P10 with conventional antibiotics against extensively drug-resistant Acinetobacter baumannii and colistin-resistant Pseudomonas aeruginosa isolates.

BACKGROUND: Infections caused by drug-resistant strains of Acinetobacter baumannii and Pseudomonas aeruginosa are now a global problem that requires the immediate development of new antimicrobial drugs. Combination therapy and using antimicrobial peptides are two strategies with high potential to solve this issue. By these strategies, this study aimed to determine the antimicrobial effect of Nisin and P10 antimicrobial peptides on extensively drug-resistant Acinetobacter baumannii and colistin-resistant Pseudomonas aeruginosa isolates, and investigate the most effective combination of an antimicrobial peptide with an antibiotic. MATERIAL AND METHODS: This study was performed on five resistant clinical isolates and one standard strain for each kind of bacterium. First, the minimum inhibitory concentrations of two antimicrobial peptides (Nisin and P10) and five common antibiotics for the treatment of Gram-negative bacteria (ceftazidime, tobramycin, ciprofloxacin, doripenem, and colistin) was determined using Scanner-Assisted Colorimetric MIC Method. Then, the combination effect of P10+Nisin, P10+antibiotics, Nisin + antibiotics was investigated using checkerboard method. RESULTS: The MIC value of Nisin and P10 against studied pathogens were 64-256 and 8-32 µg/ml, respectively. P10+Nisin combination showed synergistic effect against standard strains and additive effect against drug-resistant clinical isolates. It was also found that the combination effect of P10+ceftazidim, P10+doripenem, and Nisin + colistin was synergistic in most cases. Nisin + tobramycin combination showed synergistic effect in exposure to standard strains, while the synergy is strain-dependent against drug-resistant clinical isolates. CONCLUSION: In conclusion, the synergism of Nisin + colistin and P10+ceftazidime/doripenem could be of great therapeutic value as antimicrobial drugs against infections caused by colistin-resistant P.aeruginosa and XDR A. baumannii.

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.

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.

Antimicrobial peptides as a promising treatment option against Acinetobacter baumannii infections.

BACKGROUND: With the increasing rate of antibiotic resistance in Acinetobacter, the World Health Organization introduced the carbapenem-resistant isolates in the priority pathogens list for which innovative new treatments are urgently needed. Antimicrobial peptides (AMPs) are one of the antimicrobial agents with high potential to produce new anti-Acinetobacter drugs. This review aims to summarize recent advances and compare AMPs with anti-Acinetobacter baumannii activity. METHODS: Active AMPs against Acinetobacter were considered, and essential features, including structure, mechanism of action, anti-A. baumannii potent, and other prominent characteristics, were investigated and compared to each other. In this regard, the Google Scholar search engine and databases of PubMed, Scopus, and Web of Science were used. RESULTS: Forty-six anti-Acinetobacter peptides were identified and classified into ten groups: Cathelicidins, Defensins, Frog AMPs, Melittin, Cecropins, Mastoparan, Histatins, Dermcidins, Tachyplesins, and computationally designed AMPs. According to the Minimum Inhibitory Concentration (MIC) reports, six peptides of Melittin, Histatin-8, Omega76, AM-CATH36, Hymenochirin, and Mastoparan have the highest anti-A. baumannii power against sensitive and antibiotic-resistant isolates. All anti-Acinetobacter peptides except Dermcidin have a net positive charge. Most of these peptides have alpha-helical structure; however, ß-sheet and other structures have been observed among them. The mechanism of action of these antimicrobial agents is divided into two categories of membrane-based and intracellular target-based attack. CONCLUSION: Evidence from this review indicates that AMPs would be likely among the main anti-A. baumannii drugs in the post-antibiotic era. Also, the application of computer science to increase anti-A. baumannii activity and reduce toxicity could be helpful.

Design and Construction of a Eukaryotic Cloning Vector Encoding the mpt51 Gene of Mycobacterium tuberculosis.

BACKGROUND: Tuberculosis (TB) is the leading cause of death by infectious diseases worldwide, and especially prevalent in developing countries. Several vaccines against TB have been developed, recently. The aim of the present study was to design and construct a cloning vector encoding Mycobacterium tuberculosis (MTB) mpt51 gene. METHODS: DNA was extracted from MTB H37Rv strain. Gene-specific primers were designed using Gene Runner software and the mpt51 gene was amplified by PCR. The amplified fragment and pcDNA3.1(+) cloning vector were both digested with restriction enzymes, the mpt51 fragment was ligated into the vector, and the Escherichia coli (E. coli) TOP10 strain were transformed by the recombinant plasmid. Positive clones were identified by colony PCR, restriction enzyme digestion, and DNA sequencing. RESULTS: The mpt51 gene was successfully cloned into pcDNA3.1(+). A 6400 bp band for the pcDNA3.1(+)/mpt51 recombinant plasmid and a 926 bp band for mpt51 were observed by colony PCR, and restriction enzyme digestion on agarose gels. The DNA sequence was 100% homologous with the mpt51 fragment of H37Rv in GenBank. CONCLUSION: In the current study, the mpt51 gene of MTB was correctly cloned into pcDNA3.1(+). The expression of this recombinant vector can be studied in eukaryotic cells. Moreover, it is possible to determine the efficacy of this vector as a DNA vaccine candidate, and to test its protective function compared to BCG in animal models in future.

Epinecidin-1, a highly potent marine antimicrobial peptide with anticancer and immunomodulatory activities.

BACKGROUND: Antibiotic-resistant pathogens are an emerging threat in this century. Epinecidin-1 is a multi-functional Antimicrobial Peptide (AMP) produced by Orange-spotted grouper (Epinephelus coioides) has been shown to have extensive potentials as an alternative for current antibiotics. Due to the huge costs for the study and the production of a new drug, if an antimicrobial peptide has other beneficial functions in addition to antimicrobial activities, it would be preferred. METHODS: In this study, properties and applications of Epinecidin-1 were investigated and addressed comprehensively. To achieve this, the Google Scholar search engine and three databases of PubMed, Scopus, and Web of Science were used. RESULTS: Epinecidin-1 is a cationic AMP with an alpha-helical structure. Seven functional usages of this peptide have been reported in the literature including antibacterial, antifungal, antiviral, antiprotozoal, anticancer, immunomodulatory, and wound healing properties. Moreover, this peptide has high potential to be used as an active ingredient in cleaning solutions as well as application in vaccine production. CONCLUSION: Due to significant antimicrobial activities tested on bacteria such as Staphylococcus aureus and Helicobacter pylori and also wound healing properties, Epi-1 has high potential to be considered as an important candidate for the production of new drugs and treatment of various infections including diabetic foot ulcer and peptic ulcer. Moreover, adjuvant-like properties of Epi-1 make it a suitable candidate for the studies related to an adjuvant. Other attractive properties such as anticancer effects have also been reported for this peptide which encourages further studies on this peptide.

Review of antimicrobial peptides with anti-Helicobacter pylori activity.

BACKGROUND: The emergence of antibiotic-resistant Helicobacter pylori strains in recent years has increased the need for finding an alternative in the post-antibiotic era. One of the fields being considered for this purpose is antimicrobial peptides. The aim of this review was to provide an obvious scheme from the studied anti-H. pylori peptides and to investigate their common features. METHOD: First, all of the antimicrobial peptides with their anti-H. pylori effects have been proved up to September 2018 were selected and their information including structure, mechanism of action, and function was reviewed. To achieve this, three databases of PubMed, Scopus, and Web of science were used. RESULTS: A total of 9 groups containing 22 antimicrobial peptides were found with demonstrated anti-H. pylori effects. The nine groups included pexiganan, tilapia piscidins, epinecidin-1, cathelicidins, defensins, bicarinalin, odorranain-HP, PGLa-AM1, and bacteriocins. Most of the antimicrobial peptides, not all, had common features such as the ability to kill antibiotic-resistant strains, having α-helical structure, being cationic, with high positive charge and isoelectric point. CONCLUSION: Antimicrobial peptides with anti-H. pylori effects have the potential to replace the antibiotics, especially in the post-antibiotic era, if a rapid and low-cost production method would be found.

Modified genome comparison method: a new approach for identification of specific targets in molecular diagnostic tests using Mycobacterium tuberculosis complex as an example.

BACKGROUND: The first step of designing any genome-based molecular diagnostic test is to find a specific target sequence. The modified genome comparison method is one of the easiest and most comprehensive ways to achieve this goal. In this study, we aimed to explain this method with the example of Mycobacterium tuberculosis complex and investigate its efficacy in a diagnostic test. METHODS: A specific target was identified using modified genome comparison method and an in-house PCR test was designed. To determine the analytical sensitivity and specificity, 10 standard specimens were used. Also, 230 specimens were used to determine the clinical sensitivity and specificity. RESULTS: The identity and query cover of our new diagnostic target (5KST) were ≥ 90% with M. tuberculosis complex. The 5KST-PCR sensitivity was 100% for smear-positive, culture-positive and 85.7% for smear-negative, culture-positive specimens. All of 100 smear-negative, culture-negative specimens were negative in 5KST-PCR (100% clinical specificity). Analytical sensitivity of 5KST-PCR was approximately 1 copy of genomic DNA per microliter. CONCLUSIONS: Modified genome comparison method is a confident way to find specific targets for use in diagnostic tests. Accordingly, the 5KST-PCR designed in this study has high sensitivity and specificity and can be replaced for conventional TB PCR tests.

Designing and Construction of a Cloning Vector Containing mpt64 Gene of Mycobacterium tuberculosis.

BACKGROUND: Tuberculosis caused by Mycobacterium tuberculosis (M. tuberculosis), remains as one of the leading causes of deaths worldwide, with nearly two million death cases annually. BCG (Bacille Calmette-Guerin) continues to be the most widely used vaccine in the world, but the protective immunity differs in different parts of the world. Accordingly, new strategies including DNA vaccines are essentially needed. This study was aimed to design and construct a cloning vector containing mpt64 gene of M. tuberculosis. MATERIALS AND METHODS: M. tuberculosis H37Rv was cultured on Lowenstein Jensen medium, and genomic DNA was extracted. The mpt64 gene was amplified by PCR using designed specific primers. After the digestion of mpt64 and pcDNA3.1 (+) by BamHI and EcoRI restriction enzymes, the mpt64 fragment was ligated into the digested vector using T4 DNA ligase enzyme. Then, the recombinant vector was transformed into competent Escherichia coli (E. coli) TOP10 strain. To confirm the colonies of transformed bacteria, antibiotic resistance, colony-PCR, restriction enzyme digestion and DNA sequencing were used. RESULTS: To confirm the clones, colony-PCR using mpt64 specific primers was performed and the fragment of 718 bp was observed by gel electrophoresis. Clones were also verified by restriction enzyme digestion using BamHI and EcoRI restriction enzymes and the 718 bp fragment was observed. Furthermore, results of DNA sequencing showed 100% homology with the mpt64 fragment of H37Rv in GenBank. CONCLUSION: In this study, the mpt64 fragment was successfully cloned in pcDNA3.1 (+) vector. This construct can be used in future studies as a DNA vaccine in animal models to induce immune system responses.

Study of Bacterial Infections Among Patients Receiving Kidney Transplant in Mashhad, Iran.

OBJECTIVES: Over the past 2 decades, significant advances have been made in the management of infections after transplant; however, transplant recipients are still at high risk of infectious complications. This study aimed to evaluate the prevalence of bacterial infections and antimicrobial resistance patterns in kidney transplant recipients. MATERIALS AND METHODS: This cross-sectional study included 356 patients who received kidney transplants, regardless of the underlying disease, from 2013 to 2015 at the Montaserieh Transplant Hospital (Mashhad, Iran). Clinical samples collected from patients were sent to the microbiology laboratory for culture processing. Typing of bacteria was conducted, and susceptibility testing was performed according to the Clinical and Laboratory Standards Institute guideline by use the of disk diffusion agar method. Data were then analyzed by SPSS software (SPSS: An IBM Company, IBM Corporation, Armonk, NY, USA) using chi-square test. RESULTS: Among 356 kidney recipients (206 men and 150 women), 115 (32.3%) received transplants from living donors and 241 (67.7%) received transplants from deceased donors. Of 356 total patients, 112 patients (31.5%) had an infection at various times after transplant. The most common gram-negative and gram-positive isolated bacteria were Escherichia coli and coagulase-negative Staphylococcus, with prevalence rates of 66.1% and 48.6%. Most of the isolates were resistant against selected antibiotics. CONCLUSIONS: Because of the high prevalence of infection among transplant patients, infection prevention should receive more attention, and antibiotic susceptibility should be determined before treatment.

Evaluation of the eukaryotic expression of mtb32C-hbha fusion gene of Mycobacterium tuberculosis in Hepatocarcinoma cell line.

BACKGROUND AND OBJECTIVES: HBHA and Mtb32C have been isolated from culture supernatants of Mycobacterium tuberculosis (M. tuberculosis) and Mycobacterium bovis (M. bovis) and their immunogenicity previously studies have been confirmed. In this study, capability of constructed vector containing two mycobacterial immunodaminant antigens (Mtb32C-HBHA), in producing new chimeric protein under the in-vitro condition was examined. MATERIALS AND METHODS: In present study Huh7.5 cells was transfected with Mtb32C-HBHA -pCDNA3.1+ recombinant vector using the calcium phosphate method and expression of chimeric protein was assessed by RT-PCR and Western blot methods. RESULTS: Results of RT-PCR and Western blot showed expression of 35.5 KD recombinant protein (Mtb32C-HBHA) in this cell line. CONCLUSION: The constructed vector can produce two highly immunogenic antigens that fusion of them to gather makes chimeric antigen with new traits. Other attempts are needed to evaluate specific properties of this new antigen such as molecular conformation modeling and immunologic characteristics in future studies.

Design and Construction of a Cloning Vector Containing the hspX Gene of Mycobacterium tuberculosis.

BACKGROUND: Tuberculosis (TB) is a major cause of death worldwide. Finding an effective vaccine against TB is the best way to control it. Several vaccines against this disease have been developed but none are completely protective. The aim of this study was to design and construct a cloning vector containing the Mycobacterium tuberculosis (M. tuberculosis) heat shock protein X (hspX). METHODS: First, an hspX fragment was amplified by PCR and cloned into plasmid pcDNA3.1(+) and recombinant vector was confirmed. RESULTS: A 435 bp hspX fragment was isolated. The fragment was 100% homologous with hspX of M. tuberculosis strain H37Rv in GenBank. CONCLUSION: In this study, the cloning vector pcDNA3.1(+), containing a 435-bp hspX fragment of M. tuberculosis, was constructed. This could be used as a DNA vaccine to induce immune responses in animal models in future studies.