scholarly journals Computational design of probes to detect bacterial genomes by multivalent binding

2020 ◽  
Vol 117 (16) ◽  
pp. 8719-8726 ◽  
Author(s):  
Tine Curk ◽  
Chris A. Brackley ◽  
James D. Farrell ◽  
Zhongyang Xing ◽  
Darshana Joshi ◽  
...  
Keyword(s):  
Genomic Dna ◽  
Bacterial Infections ◽  
Bacterial Genome ◽  
Computational Design ◽  
Diagnostic Methods ◽  
Detection Sensitivity ◽  
Target Sequence ◽  
Inappropriate Use ◽  
Single Target ◽  
Target Dna

Rapid methods for diagnosis of bacterial infections are urgently needed to reduce inappropriate use of antibiotics, which contributes to antimicrobial resistance. In many rapid diagnostic methods, DNA oligonucleotide probes, attached to a surface, bind to specific nucleotide sequences in the DNA of a target pathogen. Typically, each probe binds to a single target sequence; i.e., target–probe binding is monovalent. Here we show using computer simulations that the detection sensitivity and specificity can be improved by designing probes that bind multivalently to the entire length of the pathogen genomic DNA, such that a given probe binds to multiple sites along the target DNA. Our results suggest that multivalent targeting of long pieces of genomic DNA can allow highly sensitive and selective binding of the target DNA, even if competing DNA in the sample also contains binding sites for the same probe sequences. Our results are robust to mild fragmentation of the bacterial genome. Our conclusions may also be relevant for DNA detection in other fields, such as disease diagnostics more broadly, environmental management, and food safety.

scholarly journals Bloodstream infection with Acinetobacter baumanii in a Plasmodium falciparum positive infant: a case report

2021 ◽  
Vol 15 (1) ◽  
Author(s):  
Charity Wiafe Akenten ◽  
Kennedy Gyau Boahen ◽  
Kwadwo Sarfo Marfo ◽  
Nimako Sarpong ◽  
Denise Dekker ◽  
...  
Keyword(s):  
Case Report ◽  
Bacterial Infections ◽  
Treatment Options ◽  
Female Child ◽  
Correct Choice ◽  
Blood Cultures ◽  
Microbial Culture ◽  
Acinetobacter Baumanii ◽  
Inappropriate Use ◽  
History Of

Abstract Background The increasing incidence of multi-antibiotic-resistant bacterial infections, coupled with the risk of co-infections in malaria-endemic regions, complicates accurate diagnosis and prolongs hospitalization, thereby increasing the total cost of illness. Further, there are challenges in making the correct choice of antibiotic treatment and duration, precipitated by a lack of access to microbial culture facilities in many hospitals in Ghana. The aim of this case report is to highlight the need for blood cultures or alternative rapid tests to be performed routinely in malaria patients, to diagnose co-infections with bacteria, especially when symptoms persist after antimalarial treatment. Case presentation A 6-month old black female child presented to the Agogo Presbyterian Hospital with fever, diarrhea, and a 3-day history of cough. A rapid diagnostic test for malaria and Malaria microscopy was positive for P. falciparum with a parasitemia of 224 parasites/μl. The patient was treated with Intravenous Artesunate, parental antibiotics (cefuroxime and gentamicin) and oral dispersible zinc tablets in addition to intravenous fluids. Blood culture yielded Acinetobacter baumanii, which was resistant to all of the third-generation antibiotics included in the susceptibility test conducted, but sensitive to ciprofloxacin and gentamicin. After augmenting treatment with intravenous ciprofloxacin, all symptoms resolved. Conclusion Even though this study cannot confirm whether the bacterial infection was nosocomial or otherwise, the case highlights the necessity to test malaria patients for possible co-infections, especially when fever persists after parasites have been cleared from the bloodstream. Bacterial blood cultures and antimicrobial susceptibility testing should be routinely performed to guide treatment options for febril illnesses in Ghana in order to reduce inappropriate use of broad-spectrum antibiotics and limit the development of antimicrobial resistance.

scholarly journals CRISPR as a Diagnostic Tool

Biomolecules ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 1162
Author(s):  
Seohyun Kim ◽  
Sangmin Ji ◽  
Hye Ran Koh
Keyword(s):  
Diagnostic Tool ◽  
Specific Binding ◽  
Diagnostic Methods ◽  
Specific Gene ◽  
Guide Rna ◽  
Engineering Technique ◽  
Target Dna ◽  
Target Binding ◽  
Genetic Engineering Technique ◽  
Disease Related Gene

Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system has recently gained growing attention as a diagnostic tool due to its capability of specific gene targeting. It consists of Cas enzymes and a guide RNA (gRNA) that can cleave the target DNA or RNA based on the sequence of the gRNA, making it an attractive genetic engineering technique. In addition to the target-specific binding and cleavage, the trans-cleavage activity was reported for some Cas proteins, including Cas12a and Cas13a, which is to cleave the surrounding single-stranded DNA or RNA upon the target binding of Cas-gRNA complex. All these activities of the CRISPR-Cas system are based on its target-specific binding, making it applied to develop diagnostic methods by detecting the disease-related gene as well as microRNAs and the genetic variations such as single nucleotide polymorphism and DNA methylation. Moreover, it can be applied to detect the non-nucleic acids target such as proteins. In this review, we cover the various CRISPR-based diagnostic methods by focusing on the activity of the CRISPR-Cas system and the form of the target. The CRISPR-based diagnostic methods without target amplification are also introduced briefly.

scholarly journals Computational Design of Antiviral RNA Interference Strategies That Resist Human Immunodeficiency Virus Escape

2005 ◽  
Vol 79 (3) ◽  
pp. 1645-1654 ◽  
Author(s):  
Joshua N. Leonard ◽  
David V. Schaffer
Keyword(s):  
Human Immunodeficiency Virus ◽  
Rna Interference ◽  
Highly Active Antiretroviral Therapy ◽  
Viral Evolution ◽  
Regulation Of Gene Expression ◽  
Computational Design ◽  
Delivery Systems ◽  
Target Sequence ◽  
Therapeutic Success ◽  
Immunodeficiency Virus

ABSTRACT Recently developed antiviral strategies based upon RNA interference (RNAi), which harnesses an innate cellular system for the targeted down-regulation of gene expression, appear highly promising and offer alternative approaches to conventional highly active antiretroviral therapy or efforts to develop an AIDS vaccine. However, RNAi is faced with several challenges that must be overcome to fully realize its promise. Specifically, it degrades target RNA in a highly sequence-specific manner and is thus susceptible to viral mutational escape, and there are also challenges in delivery systems to induce RNAi. To aid in the development of anti-human immunodeficiency virus (anti-HIV) RNAi therapies, we have developed a novel stochastic computational model that simulates in molecular-level detail the propagation of an HIV infection in cells expressing RNAi. The model provides quantitative predictions on how targeting multiple locations in the HIV genome, while keeping the overall RNAi strength constant, significantly improves efficacy. Furthermore, it demonstrates that delivery systems must be highly efficient to preclude leaving reservoirs of unprotected cells where the virus can propagate, mutate, and eventually overwhelm the entire system. It also predicts how therapeutic success depends upon a relationship between RNAi strength and delivery efficiency and uniformity. Finally, targeting an essential viral element, in this case the HIV TAR region, can be highly successful if the RNAi target sequence is correctly selected. In addition to providing specific predictions for how to optimize a clinical therapy, this system may also serve as a future tool for investigating more fundamental questions of viral evolution.

scholarly journals Recent Development of Rapid Antimicrobial Susceptibility Testing Methods through Metabolic Profiling of Bacteria

Antibiotics ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 311
Author(s):  
Chen Chen ◽  
Weili Hong
Keyword(s):  
Antimicrobial Susceptibility ◽  
Metabolic Profiling ◽  
Bacterial Infections ◽  
Susceptibility Testing ◽  
Antimicrobial Susceptibility Testing ◽  
Bacterial Metabolism ◽  
Resistant Bacteria ◽  
Bacterial Cells ◽  
Inappropriate Use ◽  
Key Factor

Due to the inappropriate use and overuse of antibiotics, the emergence and spread of antibiotic-resistant bacteria are increasing and have become a major threat to human health. A key factor in the treatment of bacterial infections and slowing down the emergence of antibiotic resistance is to perform antimicrobial susceptibility testing (AST) of infecting bacteria rapidly to prescribe appropriate drugs and reduce the use of broad-spectrum antibiotics. Current phenotypic AST methods based on the detection of bacterial growth are generally reliable but are too slow. There is an urgent need for new methods that can perform AST rapidly. Bacterial metabolism is a fast process, as bacterial cells double about every 20 to 30 min for fast-growing species. Moreover, bacterial metabolism has shown to be related to drug resistance, so a comparison of differences in microbial metabolic processes in the presence or absence of antimicrobials provides an alternative approach to traditional culture for faster AST. In this review, we summarize recent developments in rapid AST methods through metabolic profiling of bacteria under antibiotic treatment.

Adaptation of gltA and ssrA assays for diversity profiling by Illumina sequencing to identify Bartonella henselae, B. clarridgeiae and B. koehlerae

2021 ◽  
Vol 70 (7) ◽  
Author(s):  
Rosemonde Isabella Power ◽  
Nichola Elisa Davies Calvani ◽  
Yaarit Nachum-Biala ◽  
Harold Salant ◽  
Shimon Harrus ◽  
...  
Keyword(s):  
Illumina Sequencing ◽  
Type Species ◽  
Bacterial Infections ◽  
Sanger Sequencing ◽  
Bartonella Henselae ◽  
Diagnostic Methods ◽  
Mixed Infections ◽  
Accurate Identification ◽  
Content Type ◽  
Link Type

Introduction. Bartonellosis is an emerging zoonotic disease caused by bacteria of the genus Bartonella . Mixed Bartonella infections are a well-documented phenomenon in mammals and their ectoparasites. The accurate identification of Bartonella species in single and mixed infections is valuable, as different Bartonella species have varying impacts on infected hosts. Gap Statement. Current diagnostic methods are inadequate at identifying the Bartonella species present in mixed infections. Aim. The aim of this study was to adopt a Next Generation Sequencing (NGS) approach using Illumina sequencing technology to identify Bartonella species and demonstrate that this approach can resolve mixed Bartonella infections. Methodology. We used Illumina PCR amplicon NGS to target the ssrA and gltA genes of Bartonella in fleas collected from cats, dogs and a hedgehog in Israel. We included artificially mixed Bartonella samples to demonstrate the ability for NGS to resolve mixed infections and we compared NGS to traditional Sanger sequencing. Results. In total, we identified 74 Ctenocephalides felis, two Ctenocephalides canis, two Pulex irritans and three Archaeopsylla e. erinacei fleas. Real-time PCR of a subset of 48 fleas revealed that twelve were positive for Bartonella , all of which were cat fleas. Sanger sequencing of the ssrA and gltA genes confirmed the presence of Bartonella henselae , Bartonella clarridgeiae and Bartonella koehlerae . Illumina NGS of ssrA and gltA amplicons further confirmed the Bartonella species identity in all 12 flea samples and unambiguously resolved the artificially mixed Bartonella samples. Conclusion. The adaptation and multiplexing of existing PCR assays for diversity profiling via NGS is a feasible approach that is superior to traditional Sanger sequencing for Bartonella speciation and resolving mixed Bartonella infections. The adaptation of other PCR primers for Illumina NGS will be useful in future studies where mixed bacterial infections may be present.

Cirrhosis and Complications of Portal Hypertension

2018 ◽  
Author(s):  
Andres Cardenas ◽  
Isabel Graupera ◽  
Elsa Sola ◽  
Pere Ginès
Keyword(s):  
Liver Transplantation ◽  
Portal Hypertension ◽  
Variceal Bleeding ◽  
Bacterial Infections ◽  
Hepatorenal Syndrome ◽  
Wide Spectrum ◽  
Clinical Manifestations ◽  
Diagnostic Methods ◽  
Decompensated Cirrhosis ◽  
Ultrasound Images

Cirrhosis is the most advanced stage of all the different types of chronic liver diseases. It is defined as a diffuse disorganization of normal hepatic structure by extensive fibrosis associated with regenerative nodules. Hepatic fibrosis is potentially reversible if the causative agent is removed. However, advanced cirrhosis leads to major alterations in the hepatic vascular bed and is usually irreversible. Cirrhosis is a progressive and severe clinical condition associated with considerable morbidity and high mortality. It leads to a wide spectrum of characteristic clinical manifestations, mainly attributable to hepatic insufficiency and portal hypertension. Major complications of portal hypertension include ascites, gastrointestinal (GI) variceal bleeding, hepatic encephalopathy (HE), renal failure, and bacterial infections. In recent years, major advances in the understanding of the natural history and pathophysiology of cirrhosis and the treatment of its complications have led to improved management, quality of life, and life expectancy of patients with this disease. Cirrhosis is also a risk factor for developing hepatocellular carcinoma (HCC). Decompensated cirrhosis carries a poor short-term prognosis; thus, orthotopic liver transplantation (OLT) should always be considered in suitable candidates. This chapter describes the epidemiology, etiology and genetic factors, pathogenesis, diagnosis, general management, and treatment of cirrhosis. Complications of cirrhosis are discussed, including ascites, spontaneous bacterial peritonitis, dilutional hyponatremia, hepatorenal syndrome, variceal bleeding, hepatopulmonary syndrome and postpulmonary hypertension, HE, and HCC. Indications and contraindications for liver transplantation are described. Figures show liver biopsy results and ultrasound images in cirrhosis from hepatitis C, a patient with tense ascites, transjugular intrahepatic portosystemic shunting (TIPS), large esophageal varices with red spots, and HCC. Tables outline the main causes of cirrhosis and the diagnostic methods for identifying them, the Child-Pugh score, diagnostic criteria for hepatorenal syndrome, grades of HE, and indications for liver transplantation.This chapter contains 6 highly rendered figures, 8 tables, 73 references.

scholarly journals Prescribing Empiric Antibiotics for Febrile Neutropenia: Compliance with Institutional Febrile Neutropenia Guidelines

Pharmacy ◽  
2018 ◽  
Vol 6 (3) ◽  
pp. 83 ◽  
Author(s):  
Doaa Naeem ◽  
Majed Alshamrani ◽  
Mohammed Aseeri ◽  
Mansoor Khan
Keyword(s):  
Febrile Neutropenia ◽  
Cancer Patients ◽  
Bacterial Infections ◽  
Cross Sectional Study ◽  
Cross Sectional ◽  
Management Guidelines ◽  
Empiric Antibiotics ◽  
Inappropriate Use ◽  
Common Diagnosis ◽  
The Mean

Background: Febrile neutropenia (FN) is an oncologic emergency which should be treated immediately with empiric antibiotics. Different institutions observe different antibiograms and use different FN management guidelines. Our center implemented FN management guidelines for adult cancer patients in 2009. Hence, we decided to assess compliance with FN management guidelines and to describe the pattern of bacterial infections. Method: We conducted a cross-sectional study on all adult cancer patients admitted with FN. Data were collected from electronic medical records between January and December 2014. Results: One hundred FN episodes met the study inclusion criteria. The mean age of the patients was 41 ± 17 years; 52% (52 patients) were women. The most common diagnosis was lymphoma (33%). In terms of compliance to institutional FN guidelines, 55% of patients received guideline non-compliant treatment. The most common non-compliant treatment was incorrect amikacin dosing in 31% of patients, followed by incorrect vancomycin dosing in 20%, incorrect piperacillin/tazobactam dosing in 19%, inappropriate use of carbapenems in 18%, and non-compliant vancomycin use in 12% of patients. Bacterial isolates were only observed in 19% of the FN episodes. Among these 19 episodes of FN, Gram-negative pathogens were predominant and were identified in 74% of the episodes, followed by Gram-positive pathogens in 16% and polymicrobial pathogens in 10%. The mean time to defervescence was 2.21 ± 2 days. Conclusion: Our study concluded that there was a high percentage of non-compliance with our institutional FN management guidelines. We recommend following appropriate empiric antibiotic doses and indications as per institutional guidelines.

scholarly journals Possibilities of tropical treatment of bacterial ENT infections

2021 ◽  
pp. 44-54
Author(s):  
P. A. Shamkina ◽  
A. A. Krivopalov ◽  
P. I. Panchenko ◽  
S. V. Ryazantsev
Keyword(s):  
Antibiotic Resistance ◽  
Otitis Media ◽  
Primary Care Physicians ◽  
Bacterial Infections ◽  
Acute Otitis Media ◽  
Medical Community ◽  
Antibacterial Drug ◽  
Upper Respiratory Tract ◽  
Inappropriate Use ◽  
Acute Rhinosinusitis

The overuse and inappropriate use of systemic antibiotics is the most serious cause of problems associated with the increasing resistance of bacterial pathogens. What served as the basis for WHO to call the XXI century “The era of antibiotic resistance”. The wide spread of resistant strains of microorganisms, the growth of severe and complicated forms of diseases leads to an increase in the frequency of unfavorable treatment outcomes. In the Russian Federation, an increase in the incidence of acute rhinosinusitis from 4.6 to 12.7 cases per 1000 population has been noted in the last decade. The incidence of acute rhinosinusitis in Europe is recorded in 6.4 ± 3.6 of all cases of visits to primary care physicians. Up to 38% of outpatients in the ENT profile suffer from various forms of otitis media, including up to 30% of acute otitis media. The most important way to overcome the global problem of antibiotic resistance, along with the delayed use of systemic antibacterial drugs initiated by the world medical community, is to switch to the active use of topical drugs with antimicrobial activity. The article provides an overview of the data of domestic and foreign literature on the properties of a topical antibacterial drug with the active ingredient hydroxymethylquinoxaline dioxide. The results of experimental work and clinical studies, proving the high efficacy and safety of the drug in the complex treatment of bacterial infections of the upper respiratory tract, have been analyzed.

scholarly journals Rapid detection of cytochrome cd1-containing nitrite reductase encoding gene nirS of denitrifying bacteria with loop-mediated isothermal amplification assay

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Xuzhi Zhang ◽  
Qianqian Yang ◽  
Qingli Zhang ◽  
Xiaoyu Jiang ◽  
Xiaochun Wang ◽  
...  
Keyword(s):  
Nitrite Reductase ◽  
Genomic Dna ◽  
Limit Of Detection ◽  
Lamp Assay ◽  
Denitrifying Bacteria ◽  
Isothermal Amplification ◽  
Target Sequence ◽  
Bacterial Cells ◽  
Loop Mediated Isothermal Amplification ◽  
Order Of Magnitude

Abstract The cytochrome cd1-containing nitrite reductase, nirS, plays an important role in biological denitrification. Consequently, investigating the presence and abundance of nirS is a commonly used approach to understand the distribution and potential activity of denitrifying bacteria, in addition to denitrifier communities. Herein, a rapid method for detecting nirS gene with loop-mediated isothermal amplification (LAMP) was developed, using Pseudomonas aeruginosa PAO1 (P. aeruginosa PAO1) as model microorganism to optimize the assay. The LAMP assay relied on a set of four primers that were designed to recognize six target sequence sites, resulting in high target specificity. The limit of detection for the LAMP assay under optimized conditions was 1.87 pg/reaction of genomic DNA, which was an order of magnitude lower than that required by conventional PCR assays. Moreover, it was validated that P. aeruginosa PAO1 cells as well as genomic DNA could be directly used as template. Only 1 h was needed from the addition of bacterial cells to the reaction to the verification of amplification success. The nirS gene of P. aeruginosa PAO1 in spiked seawater samples could be detected with both DNA-template based LAMP assay and cell-template based LAMP assay, demonstrating the practicality of in-field use.

scholarly journals Prenatal diagnosis of pyruvate kinase deficiency

Blood ◽  
1994 ◽  
Vol 84 (7) ◽  
pp. 2354-2356 ◽  
Author(s):  
L Baronciani ◽  
E Beutler
Keyword(s):  
Pyruvate Kinase ◽  
Genomic Dna ◽  
Direct Method ◽  
Pcr Amplification ◽  
Prenatal Testing ◽  
Restriction Endonuclease Analysis ◽  
Diagnostic Methods ◽  
Pyruvate Kinase Deficiency ◽  
A Direct Method ◽  
Endonuclease Analysis

Abstract Prenatal testing for pyruvate kinase deficiency is often requested by parents who already have an affected child. However, before the development of molecular biologic techniques there were no suitable diagnostic methods. We present here two cases in which the diagnosis was established, one using amniotic fluid cells, the other cord blood. Two different approaches were used. The first, using a direct method of PCR amplification and restriction endonuclease analysis, detected mutations in fetus genomic DNA. The second method, using two polymorphic sites linked to the PKRL gene, enabled us to establish which chromosome had been inherited from each parent.

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