Prospective Application of Aptamer-based Assays and Therapeutics in Bloodstream Infections

Sepsis is still a severe health problem worldwide with high morbidity and mortality. Blood bacterial culture remains the gold standard for the detection of pathogenic bacteria in bloodstream infections, but it is time-consuming, and both the sophisticated equipment and well-trained personnel are required. Immunoassays and genetic diagnosis are expensive and limited to specificity and sensitivity. Aptamers are single-stranded deoxyribonucleic acid (ssDNA) and ribonucleic acid (RNA) oligonucleotide or peptide sequence generated in vitro based on the binding affinity of aptamer-target by a process known as Systematic Evolution of Ligands by Exponential Enrichment (SELEX). By taking several advantages over monoclonal antibodies and other conventional small-molecule therapeutics, such as high specificity and affinity, negligible batch-to-batch variation, flexible modification and production, thermal stability, low immunogenicity and lack of toxicity, aptamers are presently becoming promising novel diagnostic and therapeutic agents. This review describes the prospective application of aptamerbased laboratory diagnostic assays and therapeutics for pathogenic bacteria and toxins in bloodstream infections.

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Pengaruh Rimpang Kunyit (Curcuma domestica, Val.) terhadap Bakteri Usus secara in vitro

FARMASIS: Jurnal Sains Farmasi ◽

10.36456/farmasis.v2i1.3543 ◽

2021 ◽

Vol 2 (1) ◽

pp. 18-24

Author(s):

Didiek Hardiyanto Soegiantoro ◽

Gregory Hope Soegiantoro ◽

Intan Selvyanti Waruwu ◽

Yanti Octavia Theressia

Keyword(s):

Antibacterial Activity ◽

Extraction Method ◽

Pathogenic Bacteria ◽

Shigella Flexneri ◽

Public Health Problem ◽

Intestinal Bacteria ◽

High Morbidity ◽

Turmeric Extract ◽

Diarrhea Disease

The use of turmeric rhizome to treat diarrhea is written in the original Indonesian medicinal manuscript. Diarrhea disease is still a public health problem in Indonesia, because of its high morbidity and mortality. The morbidity survey conducted by Indonesian Ministry of Health shows an increasing incidence trend. One of the causes of diarrhea is an uncontrolled increase in the number of intestinal bacteria and infection by intestinal pathogenic bacteria. This study aims to determine the effect of the turmeric rhizome preparation process, both traditionally and by extraction method by maceration and soxhletation on antibacterial activity, especially intestinal bacteria, so that it can be applied by the traditional medicine industry as well as traditional herbal medicine sellers (“jamu gendong”). The research method used was to test the antibacterial activity of fresh turmeric juice, pre-dried turmeric juice, turmeric extract by maceration using 95% ethanol, and turmeric extract by soxhletation at 100°C using 95% ethanol. The intestinal bacteria used in this study were Escherichia coli, Yersinia enterolitica, Vibrio nonagglutinable, and Shigella flexneri. The results of this study indicate that the treatment process using the traditional method, both fresh turmeric juice and pre-dried turmeric juice, does not show any antibacterial activity. Turmeric extract by maceration showed antibacterial activity against all bacterias and the greatest against Vibrio nonagglutinable bacteria. Turmeric extract by soxhletation showed antibacterial activity against all bacterias and the greatest against Vibrio nonagglutinable bacteria. The conclusion of this study is that the most appropriate method used to process turmeric rhizome as a medicine for diarrhea caused by bacteria is the extraction method by maceration or soxhletation. The greatest antibacterial effect is against the Vibrio nonagglutinable bacteria.

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Polymerase Chain Reaction (PCR): A Short Review

Anwer Khan Modern Medical College Journal ◽

10.3329/akmmcj.v4i1.13682 ◽

2013 ◽

Vol 4 (1) ◽

pp. 30-36 ◽

Cited By ~ 18

Author(s):

Md Tahminur Rahman ◽

Muhammed Salah Uddin ◽

Razia Sultana ◽

Arumina Moue ◽

Muntahina Setu

Keyword(s):

Correct Diagnosis ◽

Medical Science ◽

Genetic Diagnosis ◽

High Specificity ◽

Short Review ◽

Laboratory Diagnosis ◽

Molecular Techniques ◽

Advantages And Disadvantages ◽

Specificity And Sensitivity ◽

Diagnostic Modalities

Diagnosis of disease now a days is mostly laboratory dependent. Due to recent advances in medical science and molecular biology, most of the diagnosis of uncommon, complicated, unusual presentation of disease has left the option of molecular diagnosis as the number one diagnostic modalities. Many molecular techniques are now being widely used throughout the world including PCR, flow cytometry, tissue microarray, different blots, and genetic diagnosis. Among these PCR is the most widely accepted, commonly used diagnostic modalities with very high specificity and sensitivity for correct diagnosis. We have reviewed the principle, application, advantages and disadvantages of PCR in laboratory diagnosis of disease. DOI: http://dx.doi.org/10.3329/akmmcj.v4i1.13682 AKMMC J 2013: 4(1): 30-36

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The Toxicity Testing of Cyanobacterial Toxins In Vivo and In Vitro by Mouse Bioassay: A Review

Mini-Reviews in Medicinal Chemistry ◽

10.2174/1389557521666211101162030 ◽

2021 ◽

Vol 21 ◽

Author(s):

Hamed Ahari ◽

Bahareh ‎ Nowruzi ◽

Amir Ali Anvar ◽

Samaneh Jafari Porzani

Keyword(s):

Toxicity Testing ◽

High Specificity ◽

Immunological Methods ◽

Water Bloom ◽

Mouse Bioassay ◽

Cell Extracts ◽

Specificity And Sensitivity ◽

Main Technique

: Different biological methods based on bioactivity are available to detect cyanotoxins, including neurotoxicity, immunological interactions, hepatotoxicity, cytotoxicity, and enzymatic activity. The mouse bioassay is the first test employed in laboratory cultures, cell extracts, and water bloom materials to detect toxins. It is also used as a traditional method to estimate the LD50. Concerning the ease of access and low cost, it is the most common method for this purpose. In this method, a sample is injected intraperitoneally into adult mice, and accordingly, they are assayed and monitored for about 24 hours for toxic symptoms. The toxin can be detected using this method from minutes to a few hours; its type, e.g., hepatotoxin, neurotoxin, etc., can also be determined. However, this method is nonspecific, fails to detect low amounts, and cannot distinguish between homologues. Although the mouse bioassay is gradually replaced with new chemical and immunological methods, it is still the main technique to detect the bioactivity and efficacy of cyanotoxins using LD50 determined based on the survival time of animals exposed to the toxin. In addition, some countries oppose animal use in toxicity studies. However, high cost, ethical considerations, low-sensitivity, non-specificity, and prolonged processes persuade researchers to employ chemical and functional analysis techniques. The qualitative and quantitative analyses, as well as high specificity and sensitivity, are among the advantages of cytotoxicity tests to investigate cyanotoxins. The present study aimed at reviewing the results obtained from in-vitro and in-vivo investigations of the mouse bioassay to detect cyanotoxins, including microcystins, cylindrospermopsin, saxitoxins, etc.

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Reflections of Fibrin Formation by In Vitro Testing

10.1055/s-0039-1687483 ◽

1979 ◽

Author(s):

G. Wilner

Keyword(s):

Gel Permeation Chromatography ◽

High Specificity ◽

Structural Data ◽

Specificity And Sensitivity ◽

Immunological Tests ◽

Great Specificity ◽

Technical Simplicity ◽

Derivatives Of

Measurement of enzymatically-modified fibrinogen, fibrin, or related proteolysie products represent a means whereby the action of several enzymes can be quantiated both in vitro and in vivo. Advances in physicochemical techniques and in immunology over the past decade permit translation of recently acquired primary structural data on fibrinogen and fibrin into sensitive and specific assays for detecting derivatives of these proteins in the circulation. Physicochemical techniques that have been used by various investigators include paracoagulation tests, affinity chromatography, gel permeation chromatography, and electrophoresis. The limitations in specificity and sensitivity of these techniques in detecting circulating fibrin(ogen) derivatives is offset by the technical simplicity of most of these procedures, and perhaps these techniques in combination may ultimately yield useful clinical tests. Immunological tests can, in theory, offer vastly improved specificity and sensitivity as compared with the above mentioned procedures. However, preparation of immune reagents of high specificity is both tedious and difficult, usually requiring multiple adsorbtion steps to harvest the desired antibody population. Our recent studies on the immunochemistry of human fibrinopeptide B suggest that by restricting the size of the immunizing antigen, one can readily obtain antibodies which are relatively. restricted as determined by isoelectric focusing experiments, and which also offer great specificity. The potential for developing new antibody and T cell orobes into workable assays for detecting circulating fibrin(ogen) derivatives remains to be exolored.

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Novel Primer Sets for Rapid Detection of Zymoseptoria tritici in Wheat

Plant Disease ◽

10.1094/pdis-02-20-0318-sc ◽

2020 ◽

pp. PDIS-02-20-0318

Author(s):

Adam Kuzdraliński ◽

Justyna Leśniowska-Nowak ◽

Michał Nowak ◽

Magdalena Kawęcka ◽

Anna Kot ◽

...

Keyword(s):

High Specificity ◽

Septoria Tritici Blotch ◽

Septoria Tritici ◽

In Vitro Tests ◽

Zymoseptoria Tritici ◽

Specificity And Sensitivity ◽

Wheat Leaf ◽

Primer Sets ◽

Species Specific

Zymoseptoria tritici is a fungal pathogen causing losses in wheat yields. Here, we present new primer sets for species-specific identification of this microorganism in wheat leaf samples using conventional PCR. Primer sets were validated in silico using tools available in genetic databases. Furthermore, in vitro tests were also carried out on 190 common wheat samples with visual symptoms of Septoria tritici blotch (STB) collected in Poland in three growing seasons (2015, 2016, 2017). The designed primer sets showed full hybridization to the available genetic resources deposited in the NCBI GenBank database, and their high specificity and sensitivity were demonstrated on wheat leaf samples and selected fungal strains.

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Acinetobacter baumannii Coordinates Urea Metabolism with Metal Import To Resist Host-Mediated Metal Limitation

mBio ◽

10.1128/mbio.01475-16 ◽

2016 ◽

Vol 7 (5) ◽

Cited By ~ 28

Author(s):

Lillian J. Juttukonda ◽

Walter J. Chazin ◽

Eric P. Skaar

Keyword(s):

Acinetobacter Baumannii ◽

Pathogenic Bacteria ◽

Bloodstream Infections ◽

Antimicrobial Activities ◽

Innate Immune ◽

Antimicrobial Protein ◽

Carboxylase Activity ◽

Metal Sequestration

ABSTRACT During infection, bacterial pathogens must adapt to a nutrient metal-limited environment that is imposed by the host. The innate immune protein calprotectin inhibits bacterial growth in vitro by chelating the divalent metal ions zinc (Zn 2+ , Zn) and manganese (Mn 2+ , Mn), but pathogenic bacteria are able to cause disease in the presence of this antimicrobial protein in vivo. One such pathogen is Acinetobacter baumannii , a Gram-negative bacterium that causes pneumonia and bloodstream infections that can be complicated by resistance to multiple antibiotics. A. baumannii inhibition by calprotectin is dependent on calprotectin Mn binding, but the mechanisms employed by A. baumannii to overcome Mn limitation have not been identified. This work demonstrates that A. baumannii coordinates transcription of an NRAMP family Mn transporter and a urea carboxylase to resist the antimicrobial activities of calprotectin. This NRAMP family transporter facilitates Mn accumulation and growth of A. baumannii in the presence of calprotectin. A. baumannii is found to utilize urea as a sole nitrogen source, and urea utilization requires the urea carboxylase encoded in an operon with the NRAMP family transporter. Moreover, urea carboxylase activity is essential for calprotectin resistance in A. baumannii . Finally, evidence is provided that this system combats calprotectin in vivo , as deletion of the transporter impairs A. baumannii fitness in a mouse model of pneumonia, and this fitness defect is modulated by the presence of calprotectin. These findings reveal that A. baumannii has evolved mechanisms to subvert host-mediated metal sequestration and they uncover a connection between metal starvation and metabolic stress. IMPORTANCE Acinetobacter baumannii is a bacterium that causes bloodstream, wound, urinary tract, and pneumonia infections, with a high disease burden in intensive care units. Treatment of A. baumannii infection is complicated by resistance to most antibiotics in use today, and resistance to last-resort therapies has become commonplace. New treatments for A. baumannii infection are desperately needed, but our current understanding of the bacterial factors required to cause infection is limited. We previously found that the abundant innate immune protein calprotectin inhibits the growth of A. baumannii by withholding essential metals . Despite this, A. baumannii is still able to infect wild-type mice, which produce calprotectin during infection. Here, we identify factors employed by A. baumannii during infection to overcome calprotectin-mediated metal sequestration. Moreover, we expose a connection between metal starvation and metabolism that may be a “chink in the armor” of A. baumannii and lead to new treatment options.

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Cellular discrimination using in vitro Raman micro spectroscopy: the role of the nucleolus

The Analyst ◽

10.1039/c5an01157d ◽

2015 ◽

Vol 140 (17) ◽

pp. 5908-5919 ◽

Cited By ~ 30

Author(s):

Z. Farhane ◽

F. Bonnier ◽

A. Casey ◽

A. Maguire ◽

L. O'Neill ◽

...

Keyword(s):

Cell Lines ◽

Cancer Cell ◽

High Specificity ◽

Cancer Cell Lines ◽

Cellular Organelle ◽

Specificity And Sensitivity

Raman micro spectroscopy is employed to discriminate between cell lines. Results show the importance of the nuclear sub-cellular organelle, the nucleoli, to differentiate between cancer cell lines with high specificity and sensitivity.

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In VivoBiocompatibility andIn VitroEfficacy of Antimicrobial Gendine-Coated Central Catheters

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00834-15 ◽

2015 ◽

Vol 59 (9) ◽

pp. 5611-5618 ◽

Cited By ~ 6

Author(s):

Mohamed A. Jamal ◽

Ray Y. Hachem ◽

Joel Rosenblatt ◽

Mark J. McArthur ◽

Edd Felix ◽

...

Keyword(s):

Pathogenic Bacteria ◽

Gentian Violet ◽

Bloodstream Infections ◽

Antimicrobial Efficacy ◽

Histopathological Analysis ◽

Jugular Veins ◽

Entire Study ◽

Content Type ◽

Central Catheters

ABSTRACTAntimicrobial peripherally inserted central catheters (PICCs) might reduce the incidence of central line-associated bloodstream infections (CLABSI). We tested the biocompatibility of a novel gendine-coated (combination of chlorhexidine [CHX] and gentian violet [GV]) PICC in a rabbit intravascular model and tested antimicrobial efficacy in comparison with commercially available minocycline/rifampin (M/R)- and CHX-treated PICCs in anin vitrobiofilm colonization model. Gendine-coated and uncoated control PICCs were inserted in the jugular veins of rabbits for 4 days. Histopathological analysis was performed at the end of the 4-day period, and circulating levels of CHX and GV in the blood were measured at different time points using liquid chromatography-mass spectrometry. The antimicrobial efficacy of the PICCs was tested following simulated intravascular indwells of 24 h and 1 week against clinical isolates of methicillin-resistantStaphylococcus aureus, vancomycin-resistant enterococci,Pseudomonas aeruginosa,Escherichia coli,Acinetobacter baumannii,Enterobacter cloacae,Candida albicans, andCandida glabrata. Rabbits implanted with gendine-coated PICCs exhibited reduced levels of thrombosis and inflammation compared to those of the rabbits with uncoated controls. No GV was detected in blood samples over the entire study period, and trace concentrations of CHX were detected. The gendine-coated PICCs completely prevented the adherence of all pathogens from 24 h to 1 week (P≤ 0.001), while M/R-treated, CHX-treated, and control PICCs did not. Gendine-coated PICCs were highly effective in preventing biofilm formation of multidrug-resistant pathogenic bacteria and fungi. Gendine-coated PICCs were biocompatible in an intravascular setting. Further, the pharmacokinetic testing established that acute systemic exposures of CHX and GV from the gendine-coated catheters were well within safe levels.

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Development of NCOVID-19 Colloidal Gold Immunochromatographic Test Strip

Advanced Emergency Medicine ◽

10.18686/aem.v10i1.182 ◽

2021 ◽

Vol 10 (1) ◽

pp. 1

Author(s):

Wenyi Liu ◽

Zhaohua Li

Keyword(s):

Colloidal Gold ◽

Low Cost ◽

Virus Detection ◽

High Specificity ◽

Accurate Method ◽

Rapid Screening ◽

Nucleic Acid Detection ◽

Specificity And Sensitivity ◽

Control Serum

<div><p>Purpose: To establish a fast, simple and accurate method and immunoassay test card for the detection of new coronavirus (nCOVID-19) antigen. Methods: In this study, colloidal gold immunochromatography technology was used to detect nCOVID-19 virus antigens through the sandwich method. At the same time, the preparation plan of colloidal gold was improved, and the application of rapid immune-diagnosis technology in other fields was developed. In this study, purified recombinant nCOVID-19 nucleocapsid protein is used as the antigen to prepare murine monoclonal antibodies. The BN02 antibody produced by the mouse is used as the detection antibody to couple with colloidal gold, forming a gold-labeled complex probe. BN9m1 is used as the coating antibody for the C-line, and ProA is used for the T-line. The polymerization of colloidal gold particles enables us to detect the new coronavirus antigen’s appearance. Thus an in vitro rapid detection kit for virus detection can be made. Results: The positive detection rate of the antigen quality control serum with this colloidal gold reagent was 100%. The specificity was 100%, and the sensitivity was 1ng/ml. Conclusion: The nCOVID-19 antigen detection reagent (colloidal gold method) developed in this research has high specificity and sensitivity, and can be used in conjunction with nucleic acid detection. As a means of detecting nCOVID-19, it can achieve qualitative and rapid screening of samples with advantage such as accuracy, repeatability, and low cost.</p></div>

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Immunoinformatic approach for identification of potential epitopes against Stenotrophomonas maltophilia: A global opportunistic pathogen.

Letters in Drug Design & Discovery ◽

10.2174/1570180817999201109202557 ◽

2020 ◽

Vol 17 ◽

Author(s):

R. B. Pragathi ◽

Shobana Sugumar

Keyword(s):

Mhc Class I ◽

Stenotrophomonas Maltophilia ◽

Pathogenic Bacteria ◽

Opportunistic Pathogen ◽

Class I ◽

High Morbidity ◽

Population Coverage ◽

Adhesion Properties

Background: Stenotrophomonas maltophilia is an aerobic, non-fermentative, gram-negative, multidrug-resistant, an opportunistic nosocomial pathogen. It is associated with high morbidity and mortality in severely immunocompromised pediatric patients, including neonates. Immunoinformatic analysis paved a new way to design epitope-based vaccines, which results in a potential immunogen at a lower cost, specific immunity, easy to produce, devoid of side effects, less time consuming than conventional vaccines. To date, there is no development of vaccines or antibody-based treatments for S. maltophilia-associated infections. Introduction: Currently, epitope-based peptide vaccines against pathogenic bacteria have grasped more attention. In our present study, we have utilized various immunoinformatic tools to find a significant epitope that interacts with the maximum number of HLA alleles and the maximum population coverage for developing a vaccine against Stenotrophomonas maltophilia. Methods: This study has incorporated an immunoinformatic based screening approach to explore potential epitope-based vaccine candidates in Stenotrophomonas maltophilia proteome. In this study, 4365 proteins of the Stenotrophomonas maltophilia K279a proteome were screened to identify potential antigens and could be used as the right candidate for the vaccine. Various immunoinformatic tools were used to predict the binding of the promiscuous epitopes with Major Histocompatibility Complex (MHC) class I molecules. Other properties such as allergenicity, physiochemical, adhesion properties, antigenicity, population coverage, epitope conservancy, and toxicity were analyzed for the predicted epitope. Results: This study helps in finding the major epitope in Stenotrophomonas infections. Hence the main objective in this research was to screen complete Stenotrophomonas maltophilia proteome to recognize putative epitope candidates for vaccine design. Using computational vaccinology, immunoinformatic tools approach, and several aspects are obligatory to be fulfilled by an epitope to consider as a vaccine candidate. Our findings were promising and showed that the predicted epitopes were non-allergenic and fulfilled the other parameters required for a suitable candidate based on specific physiochemical, antigenic, and adhesion properties. Conclusion: The epitopes LLFVLCWPL and KSGEGKCGA have shown the highest binding score of −103 and −78.1 kcal/mol with HLA-A*0201 and HLA-B*0702 MHC class I allele, respectively. They were also predicted to be immunogenic and non-allergen. Further various immunological tests both in vivo and in vitro methods to be performed for finding the efficiency of the predicted epitope in the development of a targeted vaccine against Stenotrophomonas maltophilia infection.

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