Improving the quality of bacteriological studies for infections caused by carbapenem resistant Klebsiella pneumoniae using molecular diagnostic methods

2020 ◽  
pp. 6-17
Author(s):  
A. Chizhikova ◽  
E. Lisitsyna
Keyword(s):  
Klebsiella Pneumoniae ◽  
Pathogenic Bacteria ◽  
Test System ◽  
Ease Of Use ◽  
Diagnostic Methods ◽  
Molecular Diagnostic ◽  
Fast Analysis ◽  
Carbapenem Resistant ◽  
Specificity And Sensitivity

The resistance of gram-negative pathogenic bacteria to antibiotics of the β-lactam group — carbapenems — is associated with the ability of bacteria to produce carbapenemases. The relevant task is to determine carbapenem-resistant microorganisms to control the spread of resistant strains and conduct effective pharmacotherapy. The results of the development of molecular diagnostic methods, including use of polymerase chain reaction (PCR), for the detection of carbapenem resistant Klebsiella pneumoniae bacteria are presented. A multiplex PCR test system was developed for detecting carbapenem-resistant K. pneumoniae producing KPC and OXA 48-like carbapenemases. The test system is characterized by high specificity and sensitivity, ease of use, fast analysis time (up to 3 hours). Its introduction into clinical and diagnostic practice is promising in terms of improving the quality of bacteriological studies.

scholarly journals Fast and Accurate Large-Scale Detection of β-Lactamase Genes Conferring Antibiotic Resistance

2015 ◽  
Vol 59 (10) ◽  
pp. 5967-5975 ◽  
Author(s):  
Jae Jin Lee ◽  
Jung Hun Lee ◽  
Dae Beom Kwon ◽  
Jeong Ho Jeon ◽  
Kwang Seung Park ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Pathogenic Bacteria ◽  
Large Scale ◽  
Diagnostic Methods ◽  
Control Measures ◽  
Clinical Isolates ◽  
Molecular Diagnostic ◽  
Resistant Bacteria ◽  
Specificity And Sensitivity ◽  
Almost All

ABSTRACTFast detection of β-lactamase (bla) genes allows improved surveillance studies and infection control measures, which can minimize the spread of antibiotic resistance. Although several molecular diagnostic methods have been developed to detect limitedblagene types, these methods have significant limitations, such as their failure to detect almost all clinically availableblagenes. We developed a fast and accurate molecular method to overcome these limitations using 62 primer pairs, which were designed through elaborate optimization processes. To verify the ability of this large-scalebladetection method (large-scaleblaFinder), assays were performed on previously reported bacterial control isolates/strains. To confirm the applicability of thelarge-scaleblaFinder, the assays were performed on unreported clinical isolates. With perfect specificity and sensitivity in 189 control isolates/strains and 403 clinical isolates, thelarge-scaleblaFinder detected almost all clinically availableblagenes. Notably, thelarge-scaleblaFinder detected 24 additional unreportedblagenes in the isolates/strains that were previously studied, suggesting that previous methods detecting only limited types ofblagenes can miss unexpectedblagenes existing in pathogenic bacteria, and our method has the ability to detect almost allblagenes existing in a clinical isolate. The ability oflarge-scaleblaFinder to detectblagenes on a large scale enables prompt application to the detection of almost allblagenes present in bacterial pathogens. The widespread use of thelarge-scaleblaFinder in the future will provide an important aid for monitoring the emergence and dissemination ofblagenes and minimizing the spread of resistant bacteria.

scholarly journals Laboratory diagnostics of tomato spotted wilt virus by PCR

2021 ◽  
Author(s):  
O.N. Morozova ◽  
D. D. Zvyagintseva ◽  
O.O. Beloshapkina
Keyword(s):  
Self Assembly ◽  
Tomato Spotted Wilt Virus ◽  
Test System ◽  
Diagnostic Methods ◽  
Molecular Diagnostic ◽  
Laboratory Diagnostics ◽  
Tomato Spotted Wilt ◽  
Specificity And Sensitivity ◽  
Wide Range ◽  
Wilt Virus

AbstractTomato spotted wilt virus is a widespread and harmful virus. It affects a wide range of host plants. The outward signs of TSWV lesions are different on different cultures. For the early diagnosis of TSWV, molecular diagnostic methods such as PCR must be used. In the course of this work, primers were developed for the diagnosis of tomato bronzing virus by real-time PCR and classical PCR. We also compared the specificity and sensitivity of various test systems for the diagnosis of tomato bronzing virus. In the course of this comparison, it was found that the self-assembly test system and the Syntol test system can be recommended for laboratory diagnostics of tomato spotted wilt virus.

A novel molecular diagnostic method for the gut content analysis of Philaenus DNA

2022 ◽  
Vol 12 (1) ◽  
Author(s):  
Isabel Rodrigues ◽  
Vítor Ramos ◽  
Jacinto Benhadi-Marín ◽  
Aránzazu Moreno ◽  
Alberto Fereres ◽  
...  
Keyword(s):  
Pathogenic Bacteria ◽  
Control Strategies ◽  
Control Measure ◽  
Coi Gene ◽  
Molecular Diagnostic ◽  
Generalist Predators ◽  
Specificity And Sensitivity ◽  
Vector Borne ◽  
Polymerase Chain ◽  
Primer Sets

AbstractPhilaenus spumarius is a vector of Xylella fastidiosa, one of the most dangerous plants pathogenic bacteria worldwide. There is currently no control measure against this pathogen. Thus, the development of vector control strategies, like generalist predators, such as spiders, could be essential to limit the spread of this vector-borne pathogen. In this study, a polymerase chain reaction (PCR)-based approach was developed to principally detect DNA of P. spumarius in the spider’s gut. Accordingly, 20 primer pairs, targeting the mitochondrial cytochrome oxidase I (COI) and cytochrome b (cytB) genes, were tested for specificity, sensitivity, and efficiency in detecting P. spumarius DNA. Overall, two primer sets, targeting COI gene (COI_Ph71F/COI_Ph941R) and the cytB gene (cytB_Ph85F/cytB_Ph635R), showed the highest specificity and sensitivity, being able to amplify 870 pb and 550 bp fragments, respectively, with P. spumarius DNA concentrations 100-fold lower than that of the DNA of non-target species. Among these two primer sets, the cytB_Ph85F/cytB_Ph635R was able to detect P. spumarius in the spider Xysticus acerbus, reaching 50% detection success 82 h after feeding. The feasibility of this primer set to detect predation of P. spumarius by spiders was confirmed in the field, where 20% of the collected spiders presented positive amplifications.

scholarly journals Multiplex logic processing isothermal diagnostic assays for an evolving virus

2018 ◽  
Author(s):  
Sanchita Bhadra ◽  
Miguel A. Saldaña ◽  
Hannah Grace Han ◽  
Grant L. Hughes ◽  
Andrew D. Ellington
Keyword(s):  
Point Of Care ◽  
Direct Analysis ◽  
Human Saliva ◽  
Ease Of Use ◽  
Molecular Diagnostic ◽  
One Pot ◽  
Diagnostic Assays ◽  
Specificity And Sensitivity ◽  
Isothermal Assay ◽  
Fail Safe

AbstractWe have developed a generalizable ‘smart molecular diagnostic’ capable of accurate point-of-care (POC) detection of variable nucleic acid targets. Our one-pot isothermal assay relies on multiplex execution of four loop-mediated isothermal amplification reactions, with primers that are degenerate and redundant, thereby increasing the breadth of targets while reducing the probability of amplification failure. An easy-to-read visual answer is computed directly by a multi-input Boolean OR gate signal transducer that uses degenerate strand exchange probes to assess any combination of amplicons. We demonstrate our platform by using the same assay to detect divergent Asian and African lineages of the evolving Zika virus (ZIKV), while maintaining selectivity against non-target viruses. Direct analysis of biological specimens proved possible, with 20 virions / µl being directly detected in human saliva within 90 minutes, and crudely macerated ZIKV-infected Aedes aegypti mosquitoes being identified with 100% specificity and sensitivity. The ease-of-use with minimal instrumentation, broad programmability, and built-in fail-safe reliability make our smart molecular diagnostic attractive for POC use.

scholarly journals Characterizing clinical carbapenem-resistant Klebsiella pneumoniae  phenotypes and genomic information reveals its molecular epidemiology

2019 ◽  
Author(s):  
Xiaoling Yu ◽  
Wen Zhang ◽  
Zhiping Zhao ◽  
Chengsong Ye ◽  
Shuyan Zhou ◽  
...  
Keyword(s):  
Klebsiella Pneumoniae ◽  
Pathogenic Bacteria ◽  
Efflux Pump ◽  
Carbapenem Resistance ◽  
Snp Markers ◽  
Nucleotide Polymorphisms ◽  
Phenotypic Resistance ◽  
Carbapenem Resistant ◽  
Oxford Nanopore ◽  
Long Read

Abstract The enhancing incidence of carbapenem-resistant Klebsiella pneumoniae (CRKP)-mediated infections in Mengchao Hepatobiliary Hospital of Fujian Medical University in 2017 promoted this investigation to study gene phenotypes and resistance genes of emergence regarding the CRKP strains. In current study, seven inpatients are enrolled in the hospital with complete treatments. The carbapenem-resistant K. pneumoniae whole genome is sequenced using MiSeq short-read and Oxford Nanopore long-read sequencing technology. Prophages are identified to assess genetic diversity within CRKP genomes. The investigation encompassed eight CRKP strains that collected from the patients enrolled as well as the environment, which illustrate that bla KPC-2 is responsible for phenotypic resistance in six CRKP strains that K . pneumoniae sequence type (ST-11) is inferred. The plasmid with IncR, ColRNAI and pMLST type with IncF[F33:A-:B-] co-exist in all ST-11 with KPC-2-producing CRKP strains. Along with carbapenemases, all K. pneumoniae strains harbor two or three extended spectrum β-lactamase (ESBL)-producing genes. F osA gene is detected amongst all the CRKP strains. The oqxA and oqxB expressions in CRKP strains may lead to carbapenem resistance since antimicrobials are expelled from pathogenic bacteria by efflux pump. The single nucleotide polymorphisms (SNP) markers are indicated and validated among all CRKP strains, providing valuable clues for distinguishing carbapenem-resistant strains from conventional K. pneumoniae .

scholarly journals Simultaneous Detection of Different Zika Virus Lineages via Molecular Computation in a Point-of-Care Assay

Viruses ◽  
2018 ◽  
Vol 10 (12) ◽  
pp. 714 ◽  
Author(s):  
Sanchita Bhadra ◽  
Miguel Saldaña ◽  
Hannah Han ◽  
Grant Hughes ◽  
Andrew Ellington
Keyword(s):  
Zika Virus ◽  
Point Of Care ◽  
Logic Gate ◽  
Simultaneous Detection ◽  
Direct Analysis ◽  
Ease Of Use ◽  
Molecular Diagnostic ◽  
Specificity And Sensitivity ◽  
Isothermal Assay ◽  
Fail Safe

We have developed a generalizable “smart molecular diagnostic” capable of accurate point-of-care (POC) detection of variable nucleic acid targets. Our isothermal assay relies on multiplex execution of four loop-mediated isothermal amplification reactions, with primers that are degenerate and redundant, thereby increasing the breadth of targets while reducing the probability of amplification failure. An easy-to-read visual answer is computed directly by a multi-input Boolean OR logic gate (gate output is true if either one or more gate inputs is true) signal transducer that uses degenerate strand exchange probes to assess any combination of amplicons. We demonstrate our methodology by using the same assay to detect divergent Asian and African lineages of the evolving Zika virus (ZIKV), while maintaining selectivity against non-target viruses. Direct analysis of biological specimens proved possible, with crudely macerated ZIKV-infected Aedes aegypti mosquitoes being identified with 100% specificity and sensitivity. The ease-of-use with minimal instrumentation, broad programmability, and built-in fail-safe reliability make our smart molecular diagnostic attractive for POC use.

Microbiological Quality of Chilled Beef Carcasses in Northern Ireland: A Baseline Survey

2001 ◽  
Vol 64 (4) ◽  
pp. 498-502 ◽  
Author(s):  
KATHRYN A. MURRAY ◽  
ARTHUR GILMOUR ◽  
ROBERT H. MADDEN
Keyword(s):  
Northern Ireland ◽  
Pathogenic Bacteria ◽  
Microbiological Quality ◽  
Viable Count ◽  
Ease Of Use ◽  
Baseline Survey ◽  
Beef Carcasses ◽  
The Mean ◽  
Yeasts And Molds

To standardize the assessment of the hygienic quality of beef carcasses in Northern Ireland (NI) abattoirs, swabbing techniques were evaluated. Six materials, including two commercially produced swabs, were compared for their ability to recover spoilage and pathogenic bacteria and for their ease of use as carcass swabs. A sponge retailed for domestic use was selected on the basis of efficiency of recovery of microorganisms, ease of use, and cost. On sample carcasses, 1,000 cm2 of the brisket was swabbed, since this site is normally readily contaminated. For 9 months, 420 carcasses in seven of the nine European Union–approved abattoirs in NI were sampled while in the chiller (24 to 48 h after kill). Total viable count (TVC), yeasts and molds, and Enterobacteriaceae were enumerated after incubation at 22 (48 h) and 37°C (48 h), and the results were expressed as log CFU/cm2. The mean TVC results at 22 and 37°C were 2.80 ± 0.70 and 2.75 ± 0.64, respectively. Although 63% of samples had yeasts that grew at 22°C, only 35% were positive at 37°C. The respective mean yeast counts were 1.12 ± 0.59 and 0.46 ± 0.51. Enterobacteriaceae were present in 15% of samples at 22°C and 21% of samples at 37°C. The mean counts for positive samples were 0.41 ± 0.37 and 0.40 ± 0.30, respectively. Molds were found in less than 4% of samples. Given that the brisket is normally one of the most heavily contaminated parts of the carcass, these results suggest that good hygienic practices are in operation in NI abattoirs. The results also enabled the abattoirs with the cleanest carcasses to be identified, hence permitting best practices to be found.

scholarly journals Genomic Epidemiology of Klebsiella pneumoniae in Italy and Novel Insights into the Origin and Global Evolution of Its Resistance to Carbapenem Antibiotics

2014 ◽  
Vol 59 (1) ◽  
pp. 389-396 ◽  
Author(s):  
Stefano Gaiarsa ◽  
Francesco Comandatore ◽  
Paolo Gaibani ◽  
Marta Corbella ◽  
Claudia Dalla Valle ◽  
...  
Keyword(s):  
Klebsiella Pneumoniae ◽  
Pathogenic Bacteria ◽  
High Morbidity ◽  
Third Generation ◽  
Data Set ◽  
Content Type ◽  
Genomic Epidemiology ◽  
Carbapenem Resistant ◽  
Resistant Group ◽  
Third Generation Cephalosporins

ABSTRACTKlebsiella pneumoniaeis at the forefront of antimicrobial resistance for Gram-negative pathogenic bacteria, as strains resistant to third-generation cephalosporins and carbapenems are widely reported. The worldwide diffusion of these strains is of great concern due to the high morbidity and mortality often associated withK. pneumoniaeinfections in nosocomial environments. We sequenced the genomes of 89K. pneumoniaestrains isolated in six Italian hospitals. Strains were selected based on antibiotypes, regardless of multilocus sequence type, to obtain a picture of the epidemiology ofK. pneumoniaein Italy. Thirty-one strains were carbapenem-resistantK. pneumoniaecarbapenemase producers, 29 were resistant to third-generation cephalosporins, and 29 were susceptible to the aforementioned antibiotics. The genomes were compared to all of the sequences available in the databases, obtaining a data set of 319 genomes spanning the known diversity ofK. pneumoniaeworldwide. Bioinformatic analyses of this global data set allowed us to construct a whole-species phylogeny, to detect patterns of antibiotic resistance distribution, and to date the differentiation between specific clades of interest. Finally, we detected an ∼1.3-Mb recombination that characterizes all of the isolates of clonal complex 258, the most widespread carbapenem-resistant group ofK. pneumoniae. The evolution of this complex was modeled, dating the newly detected and the previously reported recombination events. The present study contributes to the understanding ofK. pneumoniaeevolution, providing novel insights into its global genomic characteristics and drawing a dated epidemiological scenario for this pathogen in Italy.

Detection of NDM-1 and VIM Genes in Carbapenem-Resistant Klebsiella pneumoniae Isolates from a Tertiary Health-Care Center in Kathmandu, Nepal

Chemotherapy ◽  
2021 ◽  
pp. 1-11
Author(s):  
Sabita Thapa ◽  
Nabaraj Adhikari ◽  
Anil Kumar Shah ◽  
Ishworiya Lamichhane ◽  
Binod Dhungel ◽  
...  
Keyword(s):  
Klebsiella Pneumoniae ◽  
Bacterial Infections ◽  
Care Center ◽  
Diffusion Method ◽  
Accurate Identification ◽  
Clinical Specimens ◽  
Health Care Center ◽  
Carbapenem Resistant ◽  
Resistant Genes ◽  
Specificity And Sensitivity

<b><i>Background:</i></b> <i>Klebsiella pneumoniae</i> is one of the leading causes of nosocomial infections. Carbapenems are used as the last resort for the treatment of multidrug resistant Gram-negative bacterial infections. In recent years, resistance to these lifesaving drugs has been increasingly reported due to the production of carbapenemase. The main objective of this study was to detect the carbapenem-resistant genes <i>bla</i><sub>NDM-1</sub> and <i>bla</i><sub>VIM</sub> in <i>K. pneumoniae</i> isolated from different clinical specimens. <b><i>Methods:</i></b> A total of 585 clinical specimens (urine, pus, sputum, blood, catheter tips, and others) from human subjects attended at Annapurna Neurological Institute and Allied Sciences, Kathmandu were obtained in the period between July 2018 and January 2019. The specimens were isolated and identified for <i>K. pneumoniae</i>. All <i>K</i>. <i>pneumoniae</i> isolates were processed for antimicrobial susceptibility testing (AST) using the disk diffusion method. The isolates were further phenotypically confirmed for carbapenemase production by the modified Hodge test (MHT) using imipenem (10 μg) and meropenem (10 μg) discs. Thus, confirmed carbapenemase-producing isolates were further screened for the production of <i>bla</i><sub>NDM-1</sub> and <i>bla</i><sub>VIM</sub> using conventional polymerase chain reaction (PCR). <b><i>Results:</i></b> Among the clinical isolates tested, culture positivity was 38.29% (224/585), and the prevalence of <i>K. pneumoniae</i> was 25.89% (58/224). On AST, <i>K. pneumoniae</i> exhibited resistance toward carbapenems including ertapenem, meropenem, and imipenem, while it showed the highest susceptibility rate against to tigecycline (93.1%; 54/58). Overall, AST detected 60.34% (35/58) carbapenem-resistant isolates, while the MHT phenotypically confirmed 51.72% (30/58) isolates as carbapenemase-producers and 48.28% (28/58) as carbapenemase nonproducers. On subsequent screening for resistant genes among carbapenemase-producers by PCR assay, 80% (24/30) and 3.33% (1/30) isolates were found to be positive for <i>bla</i><sub>NDM-1</sub> and <i>bla</i><sub>VIM</sub>, respectively. In the same assay among 28 carbapenem nonproducing isolates, 9 (32.14%) isolates were positive for <i>bla</i><sub>NDM-1</sub> gene while none of them were tested positive for <i>bla</i><sub>VIM</sub> gene. <b><i>Conclusions:</i></b> Molecular detection of resistant genes provides greater specificity and sensitivity than those with conventional techniques, thus aiding in accurate identification of antimicrobial resistance and clinical management of the disease.

scholarly journals Molecular Characterization of Diarrheagenic Bacteria Isolated from Stool of Under-five Children in Dar Es Salaam. Tanzania

2015 ◽  
Vol 7 (1) ◽  
pp. 71
Author(s):  
Benjamin Enos Ngoso ◽  
Lucy Andrew Namkinga ◽  
Gamba Nkwengulila
Keyword(s):  
Pathogenic Bacteria ◽  
Dar Es Salaam ◽  
Diagnostic Methods ◽  
Molecular Diagnostic ◽  
District Hospitals ◽  
Causative Agents ◽  
Stool Samples ◽  
Dar Es Salaam City ◽  
And Control ◽  
Diarrhea Disease

<p class="jbls"><span lang="EN-GB">Diarrhea is a daily public health song in developing countries like Tanzania. The causative agents are theoretically known almost to everybody. However, the eradication of this killer disease for the under-fives is an enigma. This study aimed to provide update advantages of molecular diagnostic versus conventional methods as regards to acute diarrhea, and to determine bacterial causes of diarrhea among children aging five years and below in Dar Es Salaam, Tanzania, using multiplex PCR technique. </span></p><p class="jbls"><span lang="EN-GB">Samples were collected from the under-fives from district hospitals in Dar Es Salaam city between June 2010 and February 2014. This included children admitted due to acute and/ or chronic diarrhea. A total of 3600 stool samples were analyzed, of which 1800 samples were from diarrhea cases and 1800 samples from normal control cases. About 1080 (60%) of the patients recruited were aged less than 3 years and 983 (54.6%) were males. Diarrheagenic bacteria were isolated and identified using conventional stool cultures then were characterized by mPCR. </span></p><p class="jbls"><span lang="EN-GB">Pathogenic bacteria were detected in 67.7% of the cases and in 20% of the controls. The pathogenic bacteria most strongly associated with diarrhea disease were diarrheagenic <em>Escherichia coli</em> (21.6% of cases, 6% of controls), <em>Shigella</em> spp. (16.1% of cases, 5% of controls) and S<em>almonellae, (</em>10.6% of cases, 3% of controls. The pathogenic bacteria were mostly from children aging from 24 months and above.<strong> </strong></span></p><p class="jbls"><span lang="EN-GB">Diarrheagenic bacteria play an important role in relation to childhood diarrhea aging from two years and above. Proper diagnostic methods, prevention and control through fostering good hygiene and sanitation to water and food should be emphasized especially to oral-faecal age.</span></p>

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